Structural parameters of cellulose produced by Acetobacter Xylinum and their variation in the course of drying of gel films

Article abstract of DOI:10.1023/A:1026356816905

The cellulose-producing power of the VKM V-800 Acetobacter xylinum strain under conditions of static culture was studied. The culture medium was optimized with the aim to increase the cellulose yield and obtain highly crystalline cellulose I with molecula

Full text of DOI:10.1023/A:1026356816905

Russian Journal of Applied Chemistry, Vol. 76, No. 6, 2003, pp. 989 996. Translated from Zhurnal Prikladnoi Khimii, Vol. 76, No. 6, 2003,
pp. 1017 1024.
Original Russian Text Copyright
Zanaveskina, Klechkovskaya, Feigin.
2003 by Baklagina, Khripunov, Tkachenko, Gladchenko, Lavrent’ev, Volkov, Nilova, Denisov, Sukhanova,
MACROMOLECULAR CHEMISTRY
AND POLYMERIC MATERIALS
Structural Parameters of Cellulose Produced
by Acetobacter Xylinum and Their Variation
in the Course of Drying of Gel Films
Yu. G. Baklagina, A. K. Khripunov, A. A. Tkachenko, S. V. Gladchenko,
V. K. Lavrent’ev, A. Ya. Volkov, V. K. Nilova, V. M. Denisov, T. E. Sukhanova,
I. S. Zanaveskina, V. V. Klechkovskaya, and L. A. Feigin
Institute of Macromolecular Compounds, Russian Academy of Sciences, St. Petersburg, Russia
St. Petersburg State University, St. Petersburg, Russia
Institute of Cytology, Russian Academy of Sciences, St. Petersburg, Russia
Institute of Crystallography, Russian Academy of Sciences, Moscow, Russia
Received March 14, 2003
Abstract The cellulose-producing power of the VKM V-800 Acetobacter xylinum strain under conditions of
static culture was studied. The culture medium was optimized with the aim to increase the cellulose yield
and obtain highly crystalline cellulose I with molecular weight of about 5 105.
A considerable progress in studies of cellulose bio-
synthesis, development of the concepts of structural
hierarchy of cellulose, and discovery of two crystal-
line modifications, CI and CI , in native cellulose
samples stimulated in the past decade active studies of
evolutionarily different celluloses on the molecular and
supramolecular levels. The structure of native cellu-
loses is extensively studied by biochemical, genetic,
physicochemical, and also theoretical methods [1 12].
of a polymeric sample. For example, in the cellulose
produced by Valonia ventricosa alga (degree of crys-
tallinity 100%) I /I = 60/40, and in the animal cellu-
lose produced by Halocynthia, with the same degree
of crystallinity, this ratio is 10/90 [12].
It has been found [9] that the degree of transforma-
tion of structures Ia and Ib during heat treatment of
cellulose samples is independent of the initial crystal-
linity. In this context, a fundamental question arises
concerning exact ultrastructural localization of each
phase. This question was answered to certain extent in
recent papers [13, 14]; the procedure used was based
on different reactivities of the I and I phases. The
conclusion of [13, 14] that the I phase is situated on
the surface of microcrystals or microfibrils was also
confirmed by the data of high-resolution atomic-force
microscopy [15].
The most success has been gained in studies of cel-
lulose produced by Valonia ventricosa alga and of
bacterial cellulose produced by Acetobacter xylinum
(CAX) [3].
Two polymorph families of crystalline cellulose I
are distinguished: that rich in the I form (algal and
bacterial celluloses) and that rich in the I form (cel-
lulose of higher plants and animal cellulose) [7 10].
Electron diffraction studies showed that, in the meta-
stable I phase, cellulose chains are packed to form
a triclinic cell (space group P1) with a single tie chain,
and under certain conditions, this phase transforms in-
to the thermodynamically stable monoclinic I struc-
ture (space group P21) with two macrochains in the
unit cell [7, 10]. The I /I ratio for celluloses of var-
ious origins forms in the course of biosynthesis and
is not directly related to the degree of crystallinity
Numerous papers published in the past five years
show that the ratio and sizes of structural modifica-
tions I and I and their mutual arrangement depend
both on the evolutionary origin of cellulose samples
and on the biosynthesis conditions, varying from one
sample to another [16].
The efforts of many research teams are aimed today
at revealing the factors responsible for variation of
the I /I ratio in the course of cellulose biosynthesis.
1070-4272/03/7606-0989 $25.00 2003 MAIK Nauka/Interperiodica

990
BAKLAGINA et al.
with dye concentration. At low dye concentrations,
the microfibril formation is disturbed. At high dye
concentrations, only mini-sheets of glucan chains are
obtained in the form of tubular cellulose; in the course
of washing or photoisomerization, this structure can
transform into a microfibril [11].
Attempts are made to affect, via the composition
of the culture medium, the capability of A. xylinum
strains to produce ribbons and their aggregates with
more perfect structural characteristics. It has been
shown [19] that addition to the culture medium of
antimetabolites, such as nalidixic acid and chloram-
phenicol, makes bacterial cells longer and, as a con-
sequence, results in production of wider ribbon ag-
gregates.
The effect of polymeric additives on the formation
of CAX microfibrils of various sizes and the mechan-
ism of crystallization of cellulose I and I in them
are discussed in [20 22]. It was found that addition
of carboxymethyl cellulose or xyloglucan to the cul-
ture medium reduces formation of the I phase, in-
creasing the I fraction; according to an electron-
microscopic examination, this is associated with a de-
crease in the width of CAX microfibrils. Similar ef-
fects have also been observed by other authors [23]
upon introduction of oligo- and polysaccharides into
the culture medium. Samples of CAX and its com-
posites have been prepared under various synthesis
conditions with addition of lignin carbohydrate com-
plexes or specific polysaccharides to the culture
medium; an X-ray diffraction study of these samples
has shown that some additives increase the content of
the I phase, and, in some cases, the crystal structure
does not change (the I phase remains) or low-crys-
talline composites are obtained [23].
Fig. 1. Scheme of the structure of linear TC of A. xylinum
bacterium, illustrating the formation of a mini-sheet from
four glucan chains and of a mini-crystal from 16 chains by
a subunit: (I) catalytic site of a glucan chain, (II) TC sub-
unit, (III) mini-sheet, (IV) mini-crystal of one TC subunit,
(V) TC triplet, (VI) linear series of Acetobacter TC triplets,
(VII) ribbon of microfibrils, (VIII) crystalline microfibril
of three TC subunits, and (IX) 10 100 microfibrils/ribbon.
Three TC subunits form a crystalline microfibril, and 10
100 subunits form a ribbon.
Particular attention is given to crystallization of bac-
terial CAX as a model system for which the I /I
ratio varies with strain and cultivation conditions (e.g.,
addition of chemicals to culture medium, variation of
temperature) [3, 4, 16].
Our goal was to reveal a correlation between the
structural organization of CAX macrochains in the
course of the syntheses, suggested in [3] (Fig. 1), and
structural parameters of cellulose macrosamples in
the course of drying of CAX gel films. Here, we report
the results of optimization of the CAX synthesis by
the VKM V-880 A. xylinum strain and the structural
features of CAX samples, which were evaluated by
X-ray diffraction analysis and dielectric spectroscopy.
The interest in fine details of biosynthesis and
structure of CAX is due to the possibility of preparing
chemically pure cellulose by an environmentally ac-
ceptable procedure and using it in diverse branches
of medicine [17] and engineering [18]. Various struc-
tural levels of CAX are discussed: the structure of the
terminal complex (TC) itself; its subunit; fine struc-
tural elements formed: mini-sheet, mini-crystal, mi-
crofibril, and ribbon [3, 11]. Brown [3] suggested
a scheme (Fig. 1) illustrating the shape and size of
a linear TC and their correlation with the microfibril
size. Each TC subunit includes 16 catalytic sites form-
ing four mini-sheets, which, in turn, form a mini-
crystal. A microfibril is formed from three TC sub-
units, and a ribbon, from 10 100 microfibrils. Ex-
periments on CAX cultivation with addition of Tino-
poal dye showed that the macrochain packing varies
EXPERIMENTAL
The cellulose synthesis by the VKM V-880 A. xy-
linum strain maintained at the Microbiology Chair,
St. Petersburg State University, was performed in
a culture medium containing aqueous solutions of
yeast extract (YE), glucose, peptone, ethanol, and
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STRUCTURAL PARAMETERS OF CELLULOSE
wort in optimized concentrations, at pH 5.9 6.0.
991
The seed was a 48-h A. xylinum culture grown in a
medium containing YE and wort (6 Baling scale) in
1 : 1 ratio, with 2 wt % glucose and 1 vol % ethanol.
Cultivation was performed at 29 30 C for 6 7 days,
after which CAX was separated and intermittently
washed with boiling 0.5 1% aqueous NaOH to re-
move A. xylinum cells. Then CAX was washed to
remove NaOH with distilled water, 0.5% acetic acid
solution, and again distilled water to neutral reaction.
The resulting CAX was stored either as a gel film (in
distilled water at 5 C) or (after autoclave sterilization)
at room temperature, or it was dried in a vacuum at
40 C and stored in the dry state. The procedure was
optimized by additive-lattice experimental design [24];
the five varied factors were concentrations of the com-
ponents of the medium, and the cellulose yield was
determined at different factor values.
X-ray diffraction patterns (DRON-2 diffractome-
ter and RKV-86 X-ray camera, Ni-filtered CuK ra-
diation) were measured for the initial CAX gel films
cleaned to remove the culture medium, and also for
the films from which a certain amount of water was
removed (before and after drying in air at room tem-
perature to the air-dry state). The dielectric properties
of CAX were measured with a TR-9701 device in the
frequency range 1 100 kHz at temperatures from 140
to 120 C. A two-electrode cell with chrome-plated
brass electrodes and Teflon insulation was used. The
measurements were performed in dry air with 30
60- m-thick film samples dried in a vacuum at 60 C.
Fig. 2. Diffraction patterns of CAX in various steps of gel
film drying. (I) Intensity and (2 ) Bragg angle; the same
for Figs. 4 6. (1) Initial gel films with the dry polymer :
water ratio of 1 : 100; (2) CAX sample squeezed to remove
50% of water; (3) sample dried at 20 C for 3 h; (4) sample
dried in a vacuum at 40 C.
saccharides [27, 28]. To determine the structural char-
acteristics of CAX synthesized by the VKM V-880
strain, we used gel films prepared in a medium con-
taining 2% glucose, 0.2% peptone, 0.3% YE, and
2 vol % ethanol. The IR absorption bands and X-ray
reflections of these samples were found in the ranges
characteristic of cellulose I. Determination of the
molecular weight of CAX by sedimentation and diffu-
sion in cadoxen gave an MSD value of 5 105. The
polydispersity index Mz /Mw determined by correla-
tion laser spectroscopy in cadoxen [26] was approxi-
mately 1.1. All these data are nicely consistent with
the CAX characteristics available from the literature
[29].
It is known that A. xylinum well develops in cul-
ture media containing glucose and some other mono-
saccharides as carbon sources, and also yeast extract
as growth factor and source of nitrogen [25]. To
determine the cellulose-producing power of the VKM
V-880 A. xylinum strain, the culture medium based
on the initial medium containing glucose, YE, and
peptone was optimized. The effect of organic addi-
tives, wort and ethanol, on the biosynthesis of cel-
lulose was elucidated [26]. Determination of the cel-
lulose yield from culture media of 25 different com-
positions showed that, at the optimal combination
of the components (7 wt % glucose, 0.3 wt % YE,
30 vol % wort, 0.3 wt % peptone, 3 vol % ethanol),
up to 50 g of air-dry cellulose is obtained from 1 m2
of the reactor surface.
Since the X-ray diffraction patterns obtained from
CAX gel films dried under various conditions differed
from those reported in [23, 30], it seemed appropriate
to study in more detail the kinetics of water removal
from the CAX gel film in the course of drying.
It has been found previously that CAX can be syn-
thesized on culture media with various cheap sources
of carbon: industrial wood hydrolyzates, peat hydroly-
zates, molasses, and other media containing mono-
The diffraction pattern of the initial gel film with
the dry polymer : water ratio of 1:100 (Fig. 2, cur-
ve 1) is characterized by a broad maximum at 2 =
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992
BAKLAGINA et al.
of the CAX films dried in a vacuum at 40 C (Fig. 2,
curve 4), the positions of these four reflections re-
mained unchanged, whereas the diffuse halo virtually
disappeared.
However, the samples dried under these conditions
still contained water molecules, as suggested by the
CAX relaxation properties determined by dielectric
spectroscopy [31]. Figure 3 shows the temperature
dependence of the dielectric loss tangent, tan = f(T),
for samples of CAX and, for comparison, cotton linter
cellulose (LC). For samples of both CAX and LC,
tan
passes through a maximum at negative temper-
aturems (about 60 C, 1 kHz). According to the con-
cept of the relaxation properties of cellulose [31 33],
this range of dielectric loss in CAX must be as-
sociated with relaxation of the dipole polarization and
mobility of primary hydroxy groups of cellulose
molecules. Above 0 C, the dielectric loss increases.
At 60 70 C, the loss decreases irrespective of the
measurement frequency (curves 1, 1 ); heating in a
vacuum enhances this trend (curves 1, 2). This fact
suggests removal of compounds increasing the elec-
trical conductivity from the sample bulk (in the given
case, this is, most probably, water [33]).
Fig. 3. Temperature dependence of the dielectric loss tan-
gent tan for (1, 1 ) CAX, (2) CAX heated to 100 C in
a vacuum in the course of measurement, and (3) dried cot-
ton linter. Measurement frequency, kHz: (1 3) 1 and (1 ) 10.
Simultaneoulsy, tan
decreases, and the maxi-
mum shifts from 60 Cmto higher temperatures. This
suggests disappearance of the plasticization effect
associated with the presence of water molecules form-
ing hydrogen bonds with primary hydroxyls. The
break-down of the hydroxyl water complexes does not
cause the peak of tan
to grow in intensity, which
m
may be due to involvement of the released OH groups
in a new system of intermolecular hydrogen bonds.
Comparison of the dependences tan = f(T) for CAX
and LC samples (Fig. 3, curves 2, 3) shows that the
maximal value of tan
at 60 C for CAX is two
three times lower than fmor LC. Since the height of the
peak at 60 C is determined by the amount of prima-
ry OH groups that can participate in thermal motion
and this motion can occur in defective areas only, it
can be presumed that bacterial cellulose, compared to
linter cellulose, is characterized by a more regular
network of hydrogen bonds and a more regular struc-
ture [31 33]. Indeed, the X-ray diffraction patterns of
CAX and LC samples showed that CAX films were
more ordered (Fig. 4a, curve 1); all the reflections of
CAX were indexed in the monoclinic system of modi-
fication I, which is a two-phase system of CI and
CI .
Fig. 4. (a) Diffraction patterns and (b) scheme of X-ray
pattern obtained at (a) perpendicular and (b) parallel in-
cidence of the X-ray beam relative to the plane of the
dry sample. Sample: (a) (1) CAX and (2) LC; (b) CAX.
20 40 . When 40 50% of the initial water is re-
moved from the gel film, clear reflections appear
against the background of this halo at 2 15 16 ,
23 , 27 , 32 34 , and 40 42 , with the broad max-
imum in the range 25 30 being preserved (Fig. 2,
curve 2). After drying this sample in air at 20 C for
2 3 h, the reflection positions and intensities changed
(Fig. 2, curve 3). A noticeable diffuse background
remained in the range 2 = 20 40 , and the reflec-
tions corresponding to d 0.532, 0.435, 0.390, and
0.259 nm became the strongest. In the X-ray patterns
Particular attention should be given to the range
2 = 13 18 in the diffraction pattern of CAX. It is
seen that the 110 reflection (2 = 15 ) of the CAX
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STRUCTURAL PARAMETERS OF CELLULOSE
film (Fig. 4a, curve 1) is considerably weaker as com-
993
pared to LC (Fig. 4a, curve 2). At the same time, the
intensity of the reflection at 2 = 16 50 [sum of (110)
and (002) reflections] is many times higher as com-
pared to LC and varies from one sample to another.
To reveal a planar texture in CAX films, we meas-
ured the X-ray patterns with the plane of the dried
sample arranged in parallel with the incident X-ray
beam. Figure 4b shows the scheme of the five major
reflections of CAX, denoted by the corresponding
numerals in Fig. 4a.
We revealed an appreciable redistribution of the
reflection positions and intensities. A reflection with
d = 0.607 nm (2 = 15 ), corresponding to the reflec-
tion from the 110 planes, is clearly seen in the equa-
torial region. A reflection in the meridional region at
d = 0.523 nm [reflection from the (002) planes] is
clearly seen against the background of a weak ring
reflection at 2 = 17 . The (110) reflection in the
equator is absent; only a weak ring reflection from
(110) planes with d = 0.532 0.535 nm is observed.
Fig. 5. Diffraction patterns of CAX samples dried (1) at
40 C and (2) lyophilically after freezing the gel film at
30 C; (3) CAX sample (ATCC 53524 strain) dried in
the course of freezing [23].
As for the other reflections, the weak third reflec-
tion at 2 = 20 40 is detected in the vicinity of the
meridional region and is undoubtedly a sum of reflec-
tions from the (102) and (012) planes of the mono-
clinic lattice with d = 0.437 and d = 0.432 nm. In the
equatorial region, there is a fairly strong fourth reflec-
tion with d = 0.389 0.392 nm, corresponding to the
reflections from the (200) planes in which hydrogen-
bonded cellulose chain molecules are arranged to form
parallel layers. Finally, the fifth reflection with
d = 0.259 0.260 nm, which is a superposition of
moderately strong reflections from the system of
planes (004), (220), (031), and (023), is clearly seen
in the meridional region of the X-ray pattern of a
CAX film taken from the end side, i.e., it character-
izes the periodicity along the polymeric chain.
[23] reveals differences in the intensities of some
reflections (Fig. 5). This may be due to different pro-
cedures of sample preparation. The films were dried
on glass supports at room temperature or at 40 C in a
vacuum; they had an axial-planar texture. The CAX
sample dried in the course of freezing [23] had an iso-
tropic polycrystalline structure (Fig. 5, curve 1). To
eliminate the texturing effect, we changed the drying
procedure: A gel film was first frozen at 30 C and
then dried lyophilically. However, after such a treat-
ment, the axial-planar texture of the CAX film was
preserved, even though to a considerably lesser extent
(Fig. 5, curve 2).
The use of isotropic polycrystalline samples for
X-ray diffraction analysis allowed resolution of reflec-
tions at 2 15 and 17 and their assignment to the I
and I modifications [23]. For the textured samples
under consideration, this is impossible. However, the
presence of the (002) reflection on the meridian of the
diffraction pattern (Fig. 4b) obtained at parallel ar-
rangement of the CAX sample plane relative to the in-
cident X-ray beam indicates that the gel film contains
an appreciable amount of the monoclinic I phase with
the parameters a = 0.801, b = 0.817, c = 1.036 nm
(macromolecular axis), and = 97 , suggested in [10].
Thus, aggregates of CAX microfibrils are arranged
in the plane of the dried film, forming an axial texture
whose axis is perpendicular to the film plane. Since
the (110) reflection (2 = 15 ) is only manifested in
the majority of samples when the X-ray beam is par-
allel to the sample surface, it can be concluded that,
in the crystallites constituting CAX ribbons, the (110)
planes of the monoclinic cell are arranged parallel to
the macrosample, i.e., an axial-planar texture arises
[34].
Comparison of the diffraction patterns of CAX
films (VKM V-880 strain) prepared in this study and
CAX samples produced by the ATCC 53524 strain
The diffraction patterns of CAX films prepared
using, in the biosynthesis step, such carbon sources
as industrial hydrolyzates of wood, peat, and liquors
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994
BAKLAGINA et al.
data [35] in combination with the X-ray diffraction
patterns for CAX of the VKM V-880 strain suggest
that, in the textured samples, the content of the I
modification exceeds that of the I modification.
The possibility of obtaining different I /I ratios
in CAX ribbon formations depending on the condi-
tions of cultivating A. xylinum was discussed in [15,
23]. These papers consider the shear stresses in thin
planar ribbon ensembles of CAX in the course of sim-
ultaneous crystallization and twisting. The ribbons are
twisted owing to rotation of bacteria during their
forward motion in a culture medium. Aggregation of
microfibrils yields wide (40 60 nm) ribbons; their
twisting gives rise to end stresses, inducing formation
of the I structure. On the contrary, the I structure
is formed in the central parts of the ribbons. If the
ribbons are less twisted or microfibrils have smaller
transverse size, the number of the stressed regions is
considerably smaller, which favors formation of the I
structure in CAX. Bowling et al. [36] observed un-
twisting of CAX ribbons during degradation of cellu-
lose chains effected by fungus cellulases. This rotary
motion was explained by lifting of bending stress and
formation of relaxing conformations.
Fig. 6. Diffraction patterns of CAX samples prepared using
different carbon sources. Medium: (1) containing wood
hydrolyzate, (2) standard [26], and (3) containing glycerol.
Presumably, in our case the CAX ribbons, orient-
ing on the surface of the culture medium, form a net-
work of hydrogen bonds via primary hydroxy groups,
which can prevent twisting of ribbon ensembles and
result in a higher content of the I structure, com-
pared to I , in textured CAX samples.
Fig. 7. Model of cellulose chain packing (projection onto
the ab plane) in the monoclinic cell. (A A ) Orientation
of ribbon microfibrils in the macrofilm plane. The arrow
shows the direction of mini-sheets in the course of bio-
synthesis.
Proceeding with the concept suggested in [4], we
propose a model of cellulose chain packing in the
projection onto the ab plane for the monoclinic cell
with two macromolecules (Fig. 7). The A1 A2 line
denotes the directing defining the orientation of mi-
crofibrillar ribbons on the surface of the culture medi-
um. Such an orientation of glucoside fragments on
the microfibril surface explains the presence of a large
amount of water molecules hydrogen-bonded with
primary hydroxyls. As seen from Fig. 7, the C6 OH
groups are arranged at the phase boundary along the
A1 A2 line; according to the theoretical calculations
[6], these groups have the tg conformation in the tri-
clinic I structure and the gt conformation in the
monoclinic structure, participating in the latter case in
the hydrogen bonding between the mini-layers. The
direction of mini-layers (this concept was suggested
in [3]; Fig. 1) is marked in the scheme with an arrow.
It is seen (Fig. 7) that the mini-layers are perpendicu-
lar to the culture medium surface.
1
2
from pulp-and-paper and fruit-and-berry productions
[27, 28] show that, depending on conditions of culti-
vating A. xylinum, the CAX produced does not al-
ways have the cellulose I structure (Fig. 6, curve 2);
in some cases, it is a mixture of the cellulose I and
cellulose II modifications (Fig. 6, curve 1). The bio-
synthesis conditions also govern the extent of micro-
fibril orientation in the plane of the CAX film. For
example, using glycerol as the carbon source, we ob-
served in CAX samples a clear axial-planar texture
(Fig. 6, curve 3).
Thus, comparison of the diffraction patterns of the
CAX gel films dried by different procedures or pre-
pared under different cultivation conditions with the
data given in [23, 30] shows that, under conditions of
our experiments, a texturing effect arises. Further-
more, the previously published electron diffraction
It is known that CAX gel films can retain from 100
to 200 g of water per gram of dry polymer, preserving
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STRUCTURAL PARAMETERS OF CELLULOSE
995
2
a high intrinsic tensile strength (up to 2 kgf mm )
[26]. The X-ray patterns suggest that the microfibrillar
ribbons oriented on the film surface act not
only as a reinforcing network but also as hydrophilic
layers capable of interacting with a large amount of
water molecules. According to electron-microscopic
data [35], the diameter of the aggregates is about
50 nm, and their length is extremely large. Microfib-
rillar aggregates occupy an insignificant part of the gel
film volume, which allows introduction depending on
the degree of film drying, of diverse systems into gel
films. For example, it has been shown that a CAX gel
film is an excellent carrier of such antiseptics as Cata-
pol and Poviargol containing silver clusters [37, 38].
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ACKNOWLEDGMENTS
The study was supported financially by the Russian
Foundation for Basic Research (project no. 01-03-
33158) and NWO (grant no. 047.009.015).
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