J. Biol. Macromol., 5(1), 3-10 (2005)

Review
Novel cellulose producing system by microorganisms
such as Acetobacter sp.
Yoshihiko Amano, Fuyu Ito, Takahisa Kanda
Faculty of Engineering, Shinshu University
4-17-1 Wakasato, Nagano 380-8553, JAPANReceived March 7, 2005; Accepted March 14, 2005
History of bacterial cellulose
Cellulose is the most abundantnatural polymer in the biosphere during
human history for industrial use such asenergy, materials, paper, clothes and foods.
Higher plants in addition to certain
microorganisms and animals mostly produce
cellulose. Some strains of Acetobacter
species produce a gelatinous membrane
called a pellicle on the surface of liquid
culture, and this membrane is composed ofpure cellulose.
It was about one century ago thatthe first scientific paper was written by
Brown in 1886 (1) on a peculiar
fermentative substance that would have been
known in many places on earth and wasparticularly popular in his country under thename of “vinegar plant”. Under pure
cultivation in carbohydrate media, it wasobserved that the whole surface of the liquid
is covered with a gelatinous membrane,
which may attain a thickness of 25 mm
under favorable circumstances. On
removing the membrane from the liquid, it isfound to be very tough, especially if an
attempt is made to tear it across its plane of
growth. From chemical analysis andvarious reactions, the substance wasconcluded without doubt to be cellulose,
although microscopy at that time only gavea picture of living bacteria embedded in atransparent structureless film.
During the following one hundredyears, a number of studies have been madeon the structure of the pellicle as well as its
production. Today, it is known that thepellicle comprises a random assembly of
fibrils, less than 130 nm wide, which arecomposed of a bundle of much finer
microfibrils that are 2 to 4 nm in diameter (2,3). It is also known that the pellicle gives afilm or sheet if it is dried. The
crystallographic form of this cellulose has
been almost the same as that of “cellulose
I,” commonly found in plant cellulose, andthe molecular orientation is parallel to thedirection of cellulose chain axis (4, 5).
Such a peculiar super-molecular
structure engineered by nature has come to
be of great interest to the present author
when compared with other synthetic
polymers that have been created to makesuper-strong fibers during the last two
decades. Bacterial cellulose has beenfound to have unique structural and physical4Bacterial cellulose production

Microfibril

Subelementary fibril

Cellulose ribbon

Terminal complex

Acetobacter xylinum cellFig.1. Schematic mechanism for cellulose ribbon formation by the aggregationof cellulose chains extruded from Acetobacter cell.
properties compared with higher plant
cellulose; it has an extremely fine fiber
network and high crystallinity, sheets ofwhich have higher Young’s moduli thanthose from plants. These novel physicalproperties would be expected to be useful inmany industrial applications. To theauthor’s knowledge, bacterial cellulose iscommonly used for only indigenous desert

egl

ORF2

bcsA

bcsB

food in the Philippines, medical pads andacoustic transducer diaphragms. Present
day culture methods do not allow mass
production of bacterial cellulose at low costs,
which is necessary for its use as acommodity material. However,understanding of the structure andmechanical properties of bacterial cellulose
is also important for industrial use.

bcsC

bcsD

β-gl

Fig. 2. Structure of cellulose synthesizing protein gene of
Acetobacter xylinum. egl: endo- β-1,4-glucanase, ORF2:
unknown, β-gl: β-glucosidase, bcsA-D: cellulose synthase
operon.

Mechanism of cellulose biosynthesis
Bacterial cellulose was extruded
through cell membrane when several
cellulose chains were synthesized bycellulose synthase that are located on inner
membrane. Cellulose synthesizing protein
complexes, called terminal complexes (TC),are aligned as straight line along the longaxis of cell as shown in Fig.1. First, TC
synthesizes several chains of cellulose,

called subelementary fibril, and then several
subelementary fibrils were associates andforms microfibrils, and finally they makecellulose ribbon (6). These protein
complexes are coded by cellulose synthase
operon, which are composed of bcsA, bcsB,
bcsC and bcsD (7,8). The domain A codedby bcsA and attached inner membrane, isthought to be the catalytic domain
synthesizing cellulose from UDP-glucose.

Y. Amano et al.

3
40

2.5
2

30

1.5

20

1
10

0.5
0

Cellulose (mg/flask)

Cellulase from cellulose producing biology
Cellulases catalyze the hydrolysis
of cellulose and are thought to be potentially
useful for the transformation of cellulose
into soluble and fermentable sugars, thus
providing a source of renewable energy andchemicals (9). Cellulose degradation
requires a complex enzyme systemcomposed of endo-1,4-β-glucanase,exo-cellobiohydrolase and β-glucosidase(10). Various reports have described the
production of cellulases bycellulose-producing Acetobacter strains.
Husemann and Werner (11) first reported
that Acetobacter xylinum ATCC12733
produces a carboxymethyl-cellulose(CMC)-hydrolyzing enzyme (CMCase;
endo-1,4-β-glucanase). Two different genes
encoding endo-1,4-β- glucanase in different
strains of A. xylinum were reported by twogroups and the amino acid sequences
encoded by these genes were entirely
different in the two strains (12,13). Standal
et al. (12) reported not only the presence ofan endo-1,4-β-glucanase activity in theculture media from several
cellulose-producing Acetobacter strains but
also the location of endo-1,4-β-glucanasegene which is present upstream of thecellulose synthesizing operon of A. xylinum
ATCC23769 (Fig.2). It is noteworthy that

β-glucosidase gene is also located on thedownstream of cellulose synthase operon.
However the role of such indigenous
cellulases in cellulose production remains
unknown. Tonouchi et al. (14) reported
that the addition of a small amount ofendo-glucanase (~3 µg/mL) enhances
bacterial cellulose production by A. xylinum.
Furthermore, the addition of a large amountof β-glucosidase (100 µg/mL) from sweetalmond also enhances bacterial cellulose
production. Husemann and Werner (10)reported that the degree of polymerization(DP) of bacterial cellulose produced by A.
xylinum ATCC12733 is decreased by acellulase released into the medium during
prolonged static culture. However,Marx-Figini and Pion (15) reported that theamount of cellulose with its weight-average
degree of polymerization (DPw) increases
linearly with cultivation time, and areproportional to the cell number in

Disaccharides (mg/mL)
Cellulase activity (unit)

Another domain B coded by bcsB
accelerated cellulose synthesis by combined
to cyclic-di-GMP. It is proposed that domain
C and D coded by bcs C and D might relate
to the aggregation of each cellulose chain
synthesized, but the primary function ofthese domain have been unknown.

5

0
0

2

4

6

8

10

12

14

Time (day)Fig. 3.
Changes in cellulase activity,
cellulose production and the amounts ofoligosaccharides during the culture of A.
xylinum ATCC23769.
Symbols: ●, disaccharides; ■, cellulose;▲, cellulase activity.

6

Bacterial cellulose production

shaking-culture of A. xylinum; at the end ofcell growth, both stop changing. Theysuggest that the decrease in the DP ofcellulose is not caused by its particular
cellulase. Therefore, the factors that
regulate the DP of bacterial cellulose
produced by Acetobacter cultures is notknown yet. Moreover, the relationshipbetween the DP of bacterial cellulose and its
physical properties has never been reported.
We have also investigated the
production of cellulase in the culture brothof A. xylinum ATCC23769 (Fig. 3). It was
found that the increases of cellulase activity,
cellulose production and the amount ofoligosaccharides are inclined to show thesimilar changes during the culture. It has
been assumed that oligosaccharides
produced in the culture broth aredegradation products of cellulose.
However in the early stage of culture in thisexperiment, about 0.2 mg/mL ofoligosaccharides were produced after 1 day
incubation even though CMCase activity
was very low and little cellulose has
produced (16). In addition, morphology

changes in bacterial cellulose produced by
Acetobacter xylinum ATCC23769 wereobserved in the presence of
β-glucodisaccharides such as gentiobioseand cellobiose. Endo-β-1,4-glucanase
activity in culture broth was higher than thatin the absence of those sugars. From theseresults, we has investigated the properties ofendo-β-1,4-glucanase produced by thisbacteria.
The property of cellulase from Acetobacter
xylinum
Endo-1,4-glucanase (AEG) from A.
xylinum could hydrolyze water-soluble
cellulose such as CMC, hydroxyethyl
cellulose (Table 1) and cellodextrin, anddecreased the viscosity of the substrate (17).
On the other hand, AEG could not produce
any soluble sugars from water-insoluble
cellulose such as Avicel and H3PO4-swollen
cotton. It is interestingly that bacterial
cellulose could not degrade by own cellulase
after ribbon formation which is crystalline
cellulose. These properties werecompletely different from endo-glucanase

Table 1. Substrate specificities of endoglucanase from A. xylinum.
Substrate
Water soluble substrate
Carboxymethyl Cellulose
Xyloglucan
Hydroxyethyl Cellulose
p-Nitrophenyl Glucoside
Water insoluble substrate
Avicel
H3PO4-swollen Avicel
H3PO4-swollen Cotton
Bacterial Cellulose

Specific activity(×10-3 unit/mg)
0.66
0.40
0.68
1.06
0
0
0
0

Y. Amano et al.

from fungi. AEG could hydrolyze
cello-oligosaccharides more than DP 5 andproduced cellobiose, cellotriose andcellotetraose, but when bacterial cellulose
was added into the reaction mixture
containing cellohexaose the hydrolysis
products disappeared in the reaction mixture.
It is suggested that AEG might havetransglycosyl activity. As AEG is, however,
inverting enzyme which belongs toglycosidase family 8, the other enzyme
produced by A. xylinum could catalyze thetransglycosyl reaction. It is proposed that
this activity might be closely related tocellulose synthesis.
Oligosaccharides produced by Acetobacter
xylinum
Some researcher have tried toinvestigate production of various
carbohydrates and cellulose production in
the culture broth of the cellulose producing
microorganism Acetobacter xylinum.
Various oligosaccharides, in addition tocellulose, have already been detected after
one-day culture of this microorganism.
These sugars and oligosaccharides increased
gradually during the culture (Fig. 3) andidentified to be rhamnose, mannose, somekinds of β-linked disaccharides such as
β-1,4-linked and β-1,6-linked glucobiose(cellobiose and gentiobiose), cellotriose andcellotetraose by thin layer chromatogram
analysis (18). Valla et al. (19) also
detected mannose, rhamnose, glucuronic
acid, glucuronyl mannnose and disaccharide
consisting of two glucose monomers inculture broth of A. xylinum. They assumed
this disaccharide was probably gentiobiose,

7

though the origin of these sugars was not
identified. HPLC analysis by ABEEconversion of β-glucodisaccharides showed
the detection of disaccharides such as
gentiobiose, cellobiose and a very little
laminaribiose. Moreover hydrolysis
products from these oligosaccharides by
β-glucosidase were mainly glucose (data notshown). It is reported that some strain of A.
xylinum also produces soluble
polysaccharide, called acetan (20) as shownin Fig.5. It is assumed that theseoligosaccharides may be enzymatic
degradation products of acetan produced
simultaneously in the culture broth since thecellulase produced by this microorganism
could not degrade bacterial cellulose
produced by itself. It is proposed that
cellulase activity is correlated closely with

G1

G2

G3
G4

St

Rh

Mn

A

B

C

D

Fig. 4. Thin layer chromatogram ofoligosaccharides produced by A. xylinum.
Symbols: S1, standard sugars; G1, glucose; G2,cellobiose; G3, cellotriose; G4, cellotetraose; Rh,
rhamnose; Mn, mannose. A〜D: fractions elutedon charcoal column chromatography with various
solvent as follows. A, 5 % EtOH; B, 10 % EtOH;
C, 15 % EtOH; D, 20 % EtOH.

8

Bacterial cellulose production

cellulose production, and more closely
related to the changes in the amount ofoligosaccharides.
In addition, when A. xylinum wascultured in the presence of
β-glucodisaccharides such as gentiobioseand cellobiose, we observed the increases ofcellulase activity and cellulose production.
It is suggested that those oligosaccharides
are the degradation products from acetan, as
enzymes prepared from culture broth couldhydrolyze acetan rather than cellulose.
Future study
The effects of β-glucodisaccharides
additives, such as cellobiose and gentiobiose,on bacterial cellulose (BC) produced by A.
xylinum ATCC23769 have been examined.
The assembly of microfibrils in the bacterial
cellulose was altered in the presence of these
additives.

Rha→ rhamnose↓α-1,3Glc↓β-1,6 → gentiobioseGlc↓α-1,4GlcA→ glucuronic acid
↓β-1,2
Man 6-O-Ac → mannose↓α-1,3(Glcβ1→4Glc)n → cello-oligosaccharide↓
6-O-AcFig.5.
Structure of acetan and its
constituent sugars produced by A. xylinum.

Bacterial cellulose produced in SH
medium containing cellobiose had larger
DPw/DPn values than that produced in thecontrol medium. On the other hand, BC
produced in SH medium containing
gentiobiose (BC-G) had smaller DPw/DPnvalues. The Young’s modulus of BC sheets
made of BC-G was about 1.6 times greaterthan that of normal BC sheets. It is
confirmed that the assembly of thinner
microfibrils result in the production ofstronger BC sheets.
Previously wereported that CMC-degrading activity in thepresence of β-glucodisaccharides additives
was higher than that in the normal medium
during early stages of growth (21).
Although further studies are required toclarity the effect of additives, it is proposedthat CMC-degrading activity is closely
related to synthesis and modification ofbacterial cellulose. It is noteworthy thatmost of cellulose producing organism
produced cellulose degrading enzyme
commomly. Therefore the role of thisenzyme for cellulose synthesis expects to beclear.

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Communicated by Hiroshi Ueno