JOURNAL OF FERMENTATIONAND BIOENGINEERING

Vol. 75, No. 1, 18-22. 1993

Production of Cellulose from Glucose by Acetobacter xylinum

SATOSHI MASAOKA, 1. TATSUHIKO OHE, 2 AND NAOKAZU SAKOTA 1
Central Laboratory, Rengo Co. Ltd., 4-1-186 Ohhiraki, Fukushima-ku, Osaka 553,1 and The Osaka Municipal
Technical Research Institute, 1-6-50 Morinomiya, Joto-ku, Osaka 536,2 Japan
Received 26 May 1992/Accepted28 October 1992

Acetobacter xyUnum IFO 13693 was selected as the best ceHuiose-producing bacterium among 41 strains be-
longing to the genus Acetobacter and Agrobacterium. Cellulose was found to be produced at the liquid surface
in static liquid cultivation. The rate of cellulose production depended proportionally on the surface-area of the
culture medium and was unaffected by the depth and volume of the medium. The optimum pH for cellulose
production was 4.0 to 6.0. Glucose, fructose and glycerol were preferred carbon sources for cellulose produc-
tion. The yield of cellulose, relative to the glucose consumed, decreased with an increase in initial glucose con-
centration, and glnconic acid accumulated at a high initial glucose concentration. The decrease in cellulose
yield could be due to some glucose being metabolized to giucouie acid. However, the accumulated giueonic
acid did not affect cellulose production. The culture conditions of the bacterium for ceUuiose production were
optimized. The maximum production rate of cellulose was 36 g/d. m2, with a yield of 100~l~ for added glucose
under the optimal conditions.

Since Brown (1) noted that Acetobacter xylinum pro-
duced cellulose at the surface of a medium, the biochemi-
cal reactions of cellulose synthesis have been extensively
documented (2-9). Cellulose is formed from glucose via
glucose-6-phosphate, giucose-l-phosphate, and uridine-
5'-diphosphate glucose. The mechanism of cellulose
microflbril formation has also been investigated actively
(4, 8, 10-17).
Cellulose produced by an Acetobacter strain was found
to be chemically pure, free of lignin and hemicellulose
(18), and to have different properties from wood-derived
cellulose (18-20). Recently, taking advantage of its pro-
perties, bacterial cellulose has been applied to practical
uses (19-21).
Though the improvement of cellulose production is es-
sential for the industrial production of bacterial cellulose,
relatively few reports have discussed in detail the relation-
ship between cellulose production and culture conditions
(21, 22).
In this study, we selected A. xylinum IFO 13693 as the
best cellulose-producing strain among 41 strains belonging
to Acetobacter and Agrobacterium, and investigated the
culture condition for cellulose production.
MATERIAL A N D METHODS
Microorganisms Twenty four strains of the genus
Acetobacter and seventeen strains of the genus Agrobacte-
rium were obtained from IFO (Institute for Fermentation
Osaka, Osaka).
Media and cultivation A standard medium which
consisted of 2% pepton (Nippon Seiyaku Co. Ltd.,
Tokyo), 0.5% yeast extract (Nippon Seiyaku Co. Ltd.,
Tokyo), 0.5°~ D-glucose, 0.1% MgSO4-7H20 and 0.2%
ethanol (pH6.0), was used for cellulose production.
Hestrin and Schramm's medium (18) was also used for
screening of the cellulose producing strain.
* Corresponding author. Present address: Central Laboratory,
Rengo Co. Ltd., 4-1-186 Ohhiraki, Fukushiraa-ku, Osaka 553,
Japan.
18
Seed cultures were prepared by inoculating cells grown
on standard medium agar slants into 500 ml Sakaguchi
flasks containing 70 ml of the standard medium, followed
by incubation at 30°C for 2 d with shaking (140 rpm). The
cells were then collected by centrifugation (13,000 rpm, for
20 min, at 10°C) and transferred into fresh standard me-
dium for cellulose production, followed by static incuba-
tion at 30°C.
Analytical methods The initial cell concentration
was estimated by measuring the optical density at 660 nm
(OD660). To measure the amount of cellulose, cellulose
was separated from the culture broth and cells with a
sieve (openings 45 pm). Cellulose was then washed with
deionized water. The washed cellulose was successively
boiled in a 2% NaOH solution for 20 min and washed with
deionized water. The cellulose was then dried overnight at
95°C and weighed after cooling to room temperature.
Glucose was measured by the glucose oxidase/peroxi-
dase method (23), using a Glucose C-Test Wako (Wako
Pure Chemical Industries, Ltd., Osaka). Gluconic acid
was measured by the method of Coffee et al. (24).
RESULTS AND DISCUSSION
Screening of the best strains for cellulose production
Some strains belonging to the genus Acetobacter and
Agrobacterium are known to produce cellulose (25).
Forty-one strains were tested for their cellulose production
by cultivation in Hestrin and Schramm' s medium for 7 d.
Four strains of Acetobacter xylinum produced significant
amounts of cellulose, but the following strains did not:
Acetobacter acetigenus (IFO3277, IFO3278, IFO3279,
IFO3280), Acetobacter pasteurianus (IFO13751,
IFO13753, IFO13754, IFO3170, IFO3191, IFO3222,
IFO3223, IFO3225), Acetobacter rancens (IFO3297,
IFO3298), Acetobacter aceti (IFO3281, IFO3283,
IFO3284), Acetobacter sp. (IFO3284), Agrobacterium
radiobacter (IFO12607, 12664, 12665, 13258, 13259,
13532, 13533), Agrobacterium rhizogenes (IFO13257,
IFO14554, 14555), Agrobacterium rubi (IFO13260,
IFO13261), Agrobacterium tumefaciens (IFO3058,
VOL. 75, 1993
TABLE 1. Screening of the best strain for cellulose production
Strain Cellulose yield (g)
A, xylinum
IFO 3288 0.010
IFO 13693 0.312
IFO 13772 0.257
IFO 13773 0.038
Each strain was cultivated in standard medium for 3 d. Initial OD~0
--0.7, V=60ml, S=54cm 2, H = I . I cm.
IFO12667, IFO13263, IFO13264, IFO13265). Table 1
shows the amount of cellulose produced after 3 d. Since
A. xylinum (IFO 13693) produced a higher amount of cel-
lulose than the other three strains, we used A . xylinum
(IFO 13693) in following studies.
Effhect of culture volume, surface-area and depth on
cellulose production Figure 1 shows the amount of
cellulose produced in cultures with different volume and
depth, and constant surface-area (100 cm2). The culture
volume and depth did not influence production.
Figure 2 shows the amount of cellulose produced in cul-
tures with different surface-area and depth, at constant
volume (300 cm3). The production increased with an in-
crease of surface-area (Fig. 2A), with the production rate
depending proportionally on the surface-area (Fig. 2B).
Position of cellulose formation in the culture medium
Cellulose has generally been described as being formed at
the liquid-air interface of the medium (1, 20, 26, 27), but
there has been no critical analysis of the position of cellu-
lose formation. We examined whether or not the cells
which e~Asted in the interior of the medium could produce
cellulose. As shown in Fig. 3, a mesh (opening size 22 pm),
positioned 2.5 cm from the base and 1.1 cm from the
air/liquid interface, was immersed for 4 d in the medium.
Cellulose formed at the interface, but not under the mesh.
This result suggests that only cells existing close to the
surface of the medium could produce cellulose.
Effects of initial p H on cellulose production The
$0.8
~0.6
-~ o.4
tp
0.2
I I t ~Sieve
0 I 2 3 4
Culture time (d)
FIG. 1. Effect of culture volume (V) and depth (H) on the produc-
tion of cellulose in glass vesselsat a constant surfaee-arca of 100cm2.
InitialODee0"~0.8. Symbols: o , v = 1 0 0 c m S H = l . 0 c m ; O , V = 2 0 0
cm3 H=2.0cm; G, V=400em 3 H=4.0cm; ~, V=600cm 3 H=6.0
cm; O, V=750cm 3 H=7.5cm.
CELLULOSE PRODUCTION BY A. XYLINUM 19
1.4
~
1.2
1.0
,~ _~o.4
0.8
o 0.6
0.4
~0.~ o. 0 20~'~'~ '~
I00 -
Culture surfa¢o (©m2)
0 t 2 3 4 5
Culture time (d)
FIG. 2. Effect of culture surface-area (S) and depth (H) on the
production of cellulose in glass vessels of various diameters. Initial
OD.,0-0.8, V=300 cm3. (A) Time courses of cellulose production at
different surface areas. Symbols: O, S=232cm 2 H=l.3cm; O,
S=125cm 2 H=2.4cm; G, S=93cm 2 H=3.2cm; ~, S=69cm z
H=4.3 cm; e, S=4A cm2 H=6.9 cm. (13)Rate of cellulose produc-
tion vs. surface area.
effect of initial pH on the cellulose production was tested
in the range pH 2.5 to 7.7. The amount o f cellulose in-
creased linearly with culture time for 3 d. The optimum
p H for cellulose production was 4.0 to 6.0 (Fig. 4). The
p H value of the medium remained within 0.7 of the initial
pH throughout the cultivation period, except in the case
of an initial pH of 7.7.
The optimum pH for in vivo cellulose production by A.
xylinum has previously been reported in several studies to
be less than 7.0 (18, 20, 21), and our results agreed with
these findings. On the other hand, Greathouse (28), using
a cell-free system, reported an optimum p H 8.0 to 9.0.
Effects of carbon source on cellulose production In
general, glucose has been used as a carbon source for cellu-
lose production by A. x),linum. It has been reported, how-
ever, that cellulose was also synthesized from other carbon
sources, such as 5- or 6- carbon monosaccharides, oligo-
saccharides, starch, alcohols and organic acids (29, 30).
We examined the relative yields of cellulose production
from various carbon sources, as shown in Table 2, Fruc-
tose and glycerol gave almost the same cellulose yield as
glucose, and the yield from D-glucono lactone was 62%
relative to glucose.
Effects of initial glucose concentration on cellulose pro-
duction The effects of the initial glucose concentration
(0-4.8 g/flask, i.e. 0--4.0%) on cellulose production are
shown in Fig. 5. A constant rate of production (36 g-cellu-
lose/d, m 2) was obtained for every initial glucose concen-
tration.
/ \
G1a s s beak e r ~ Cellulose pellicle
[.~.\\x \ x x \ \ \ \ \, ~t/~/blesh(openingsize = 22 /zm)
FIG. 3. Schematic diagram of experiment to check the position
of cellulose production. Initial ODs~=0.73, V--200ml, S=56cm 2,
H=3.6cm.
20 MASAOKA ET AL. J. FERMENT.BIOENO.,

0.10 •,- 5.0

O.

4.0
-~O 0.08~-
( ' '. . . . . A r,..o
0.06~ 4.5-\e
/
~f .4 4~ - 0" - 3.5
0.0 2.i 1 1~,'"
= 3.0
"~ 0.02

®
02 6 7
Initial pH a / . . ,, °
FIG. 4. Effect of initial pH on the production of cellulose. The
production rate of cellulose was calculated with the amount of cellu- ,.0- \ ,.5
lose produced in 3 d culture. Initial O D ~ - 0 . 8 , V=60 ml, S=54 cm2,
H = 1.1 cm.

The yield o f cellulose to consumed glucose, however,

decreased with an increase in initial glucose concentra-
tion. The yields o f cellulose at initial glucose concentra-
tions o f 0.6, 1.2, 2.4 a n d 4.8 g/flask were 100, 100, 68
a n d 28% respectively. In order to determine the reason Culture time (d)
for the decrease in cellulose yield, the c o n s u m p t i o n o f glu- FIG. 5. Effect of initial glucose concentration on the production
cose, accumulation o f gluconic acid, a n d change in p H of cellulose. Initial OD6~-0.8, V= 120 ml, S=66 cm2, H = 1.8 cm.
were measured during cultivation. The initial glucose concentrations were 0.6 ( [] ), 1.2 (©), 2.4 (®) and
Figure 5A shows the relationship a m o n g the c o n s u m p - 4.8 ( e ) g/flask. (A) Relationship among production of cellulose,
tion o f glucose, accumulation o f gluconic acid, a n d pro- consumption of glucose and accumulation of gluconic acid. Lines:
- - , cellulose; . . . . , glucose; .... , gluconic acid. (13) Time course of
pH.
TABLE 2. Effect of various carbon sources on the production
of cellulose
duction o f cellulose. Trace a m o u n t s o f gluconic acid were
Carbon source Cellulose yield (relative ~ ) detected in the m e d i u m when the initial glucose concentra-
Monosaccharides D-fructose 92 tions were 0.6 and 1.2 g/flask. W h e n the initial glucose
D-galactose 15 concentration was 2.4 g/flask, 0.4 g o f gluconic acid was
D-glucose 100 t e m p o r a r i l y accumulated, b u t this disappeared after
D-mannose 3 6 d. W h e n the initial glucose concentration was 4.8 g/flask,
D-xylose I1 the a m o u n t o f gluconic acid increased during the cultiva-
L-arabinose 14
L-sorbose 11 tion period.
A t initial glucose concentrations o f 2.4 a n d 4.8 g/flask,
Disaccharides lactose 16 the total a m o u n t o f cellulose and gluconic acid p r o d u c e d
maltose 7 corresponded to the a m o u n t o f glucose consumed in the
sucrose 33 period during which gluconic acid was increasing. This sug-
Polysaccharide starch 18 gests that glucose not used for cellulose synthesis is metab-
Alcohols ethanol 4 olized via gluconic acid to other substances.
ethylene glycol 1 W h e n the initial glucose concentration was less than
diethylene glycol 1 2.4 g/flask, the p H value in the m e d i u m varied within the
propylene glycol 8 range 4 to 6.4 (Fig. 5B). These changes m a y be a result o f
glycerol 93 gluconic acid formation. However, they do not correlate
myo-inositol 17 with the decrease in cellulose yield, because the o p t i m u m
Organic acids citric acid 20 p H range for cellulose p r o d u c t i o n was 4.0 to 6.0, as shown
L-malic acid 15 in Fig. 4. W h e n the initial glucose concentration was
succinic acid 12 4.8 g/flask, the p H value decreased to 3.0. Therefore, the
cellulose yield was lower than that at an initial glucose
Other D-glucono lactone 62
concentration o f 2.4 g/flask. S c h r a m m et al. (3) previously
No cabon source 2 stated that washed cells o f A. xylinum oxidized a p o r t i o n
Cells were cultivated for 3 d in the standard medium with an initial o f glucose a d d e d to gluconic acid, a n d the accumulated
carbon source concentration of 0.3 g/flask. The yield of cellulose (%) gluconic acid lowered the p H o f the culture m e d i u m and
was calculated relative to the yield in D-glUCOSemedium. Initial OD~o inhibited cellulose p r o d u c t i o n (31). O u r results agreed with
--0.8, V : 6 0 m l , S=54cm z, H : I . I era. these findings at a high glucose concentration. It is inter-
VOL. 75, 1993
(~A th.
1.5
'M
,, • -I ¢-
_o
o
o ~5
I I I ~IIS. _,
0 4 8 12 16 (I)
Culture time ( d )
FIG. 6. Effect of gluconic acid on the production of cellulose.
Initial glucose concentration=2.4g/flask, initial OD~0-0.8,V=
120ml, S=66cm 2, H = 1.8 cm. The initial gluconic acid concentra-
tions were 0Yo (o), 0.1% ( a ), and 0.3°~ (A). Lines: - - , cellulose; .... ,
glucose.
esting that the metabolism o f glucose to gluconic acid was
stimulated at an initial glucose concentration o f 4.8 g/flask
c o m p a r e d to the m e t a b o l i s m at 2.4 g/flask.
Effects o f glucoule acid on cellulose p r o d u c t i o n As
described above, gluconic acid was accumulated in the me-
d i u m b y A . xylinum. A d d i t i o n o f gluconic acid, however,
did not affect the cellulose p r o d u c t i o n (Fig. 6). Conse-
quently, the decreases in cellulose yield shown in Fig.5 are
ascribed to the partial metabolism o f glucose to gluconic
acid. These results suggest that a m u t a n t o f A . xylinum
deficient in glucose dehydrogenase activity might be m o r e
efficient for cellulose p r o d u c t i o n in a m e d i u m with glucose
as the sole c a r b o n source.
In this study, the culture conditions o f a bacterium used
for cellulose p r o d u c t i o n were optimized, giving a maxi-
m u m cellulose p r o d u c t i o n rate o f 36 g / d . m 2, with a yield
o f 100% o f a d d e d glucose. However, since the batch-type
static culture m e t h o d is used for bacterial cellulose pro-
duction, an e n o r m o u s bioreactor would be required for
industrial-scale p r o d u c t i o n . W e will continue our inves-
tigations with the aim o f solving the problems associated
with the industrial p r o d u c t i o n o f bacterial cellulose.
ACKNOWLEDGMENT
We are grateful to Y. Nara for her technical assistance.
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