Separation and Purification Technology 27 (2002) 163– 171

www.elsevier.com/locate/seppur

Pervaporation of binary water–ethanol mixtures through

bacterial cellulose membrane
Vinita Dubey a,*,1, Chhaya Saxena a, Lokendra Singh a, K.V. Ramana b,
R.S. Chauhan a
a
Defence Research & De6elopment Establishment, Jhansi Road, Gwalior, MP 474002, India
b
Defence Food Research Laboratory, Mysore, India
Received 2 August 2001; received in revised form 15 November 2001; accepted 27 November 2001

Abstract

Cellulose membrane produced by the bacterium Acetobacter xylinum was deproteinated and investigated for
pervaporation (PV) of binary water–ethanol mixtures. The membrane was characterised using elemental analysis,
infra-red spectroscopy, scanning electron microscopy as well as for mechanical strength and sorption characteristics.
A batch stirred cell was used to study the PV behaviour of water– ethanol mixtures through the membrane. The
permeate flux, selectivity, PV separation index (PSI), solubility and degree of sorption were studied as a function of
increasing ethanol concentration in the feed. The membrane was found highly selective to water. Even when the feed
was rich in ethanol ( \70% (w/w)) the permeate contained higher than 95% (w/w) of water. For feed compositions
containing less than 30% water, the selectivity towards water was in the range of 125– 287, the flux was greater than
100 g/m2 h and PSI was of the order of 104 g/m2 h. © 2002 Elsevier Science B.V. All rights reserved.

Keywords: Bacterial cellulose membrane; Pervaporation; Water– ethanol mixtures; Flux; Selectivity; Acetobacter xylinum

1. Introduction ‘bacterial cellulose’. The cellulose of bacterial

Acetobacter xylinum, a rod shaped, aerobic
gram negative bacterium which occurs as a con-
taminant in vinegar production produces a white
gelatinous material (pellicle) on the surface of the
liquid medium in a static culture system at 30 –
40 °C. In 1886, Brown first reported [1] that the
pellicle was composed of pure cellulose called
* Corresponding author. Fax: + 91-751-341148.
E-mail address: syn@drde.8m.com (V. Dubey).
1
india – dv@yahoo.com.
origin is distinguished by a high degree of crys-
tallinity and superior mechanical properties. Its
unique structural features and properties facilitate
diverse applications, ranging from wound-dress-
ing, carrier for mammalian cell culture, immobi-
lization of enzymes and other biomolecules, to
diaphragms in speakers for audio-communication
[1–3].
The pellicle which is flat can be easily processed
into a porous membrane possessing good mechan-
ical strength, unlike the cellulose from plant
biomass. Plant cellulose is often interspersed with

1383-5866/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved.
PII: S 1 3 8 3 - 5 8 6 6 ( 0 1 ) 0 0 2 1 0 - 6
164 V. Dubey et al. / Separation and Purification Technology 27 (2002) 163–171
lignin, hemicellulose and pectin leading to non-
uniformity in porosity and unpredicatable perme-
ability. In contrast the porosity of membranes
from bacterial cellulose can be suitably tailored by
varying the physiological conditions of bacterial
growth such as composition of the culture media,
its pH, temperature, oxygen tension as well as by
chemical modifications [3–6]. In contrast to poly-
mer membranes, the bacterial cellulose membrane
(BCM) can be economically produced and pro-
cessed, it also has high resistance to corrosive
chemicals and is biodegradable too, and hence
ecofriendly. These unique features and the non-
availability of any reports on use of BCM for
chemical separations prompted us to investigate
its pervaporation (PV) characteristics.
PV is a membrane based separation process in
which the membrane functions as a selective bar-
rier for the mixture to be separated. Low energy
consumption and mild working conditions make
the process attractive for separating azeotropic
and close-boiling mixtures or dehydrating temper-
ature-sensitive products. Pervaporative dehydra-
tion of ethanol has been widely studied using
membranes based on poly(vinyl alcohol),
polyamides, polysulfonamides, poly(ethyleneimi-
ne) polysiloxanes [7– 10], etc. Chitosan and its
derivatives as well as sodium alginate have also
been used for water– ethanol separations [11,12].
The potential of BCM in the dehydration of
alcohol, especially in the azeotropic mixtures, is
shown in the present study. The effect of varying
ethanol concentrations in the feed, on the perme-
ate composition and flux, selectivity, sorption and
PV separation index (PSI) has been investigated.
2. Experimental
2.1. Materials
BCM from A. xylinum was grown in Biotech-
nology Division of DRDE, Gwalior. The chemi-
cals used in the preparation of culture medium
were reagent grade obtained from M/s Fluka, E.
Merck or Aldrich. Absolute ethanol (E. Merck,
HPLC grade) and deionized distilled water were
used to prepare the binary mixtures.
2.2. Preparation of inoculum and membranes
Acetobacter culture was maintained on sucrose
medium. The composition of the medium is given
in Table 1. The pH of the medium was adjusted at
5.0 using 1.0 M HCl. The inoculum was kept for
2 weeks at 35 °C in a BOD incubator. The pelli-
cle was gently removed from the flask, thoroughly
washed with distilled water several times and
pressed to form a membrane. The membrane was
then allowed to dry at ambient temperature, and
was referred to as nascent or untreated mem-
brane. These membranes are largely composed of
proteins and cellulose. Some of these membranes
were dipped for 48 h, in a flat-bottomed large
petridish containing saturated solution of NaOH.
Since NaOH solubilises the proteinaceous matter
from the membrane, only a cellulosic matrix re-
mains in the membrane. NaOH treated mem-
branes are, therefore, also termed as
deproteinated membranes. These membranes were
rinsed several times with distilled water, until a
neutral pH was attained in the drained liquid. The
membranes were dried for a week at ambient
conditions, prior to use.
2.3. Characterisation of membranes
The elemental analysis and the infra-red spectra
(Perkin–Elmer FTIR) of the membranes were
recorded before and after the alkali treatment.
Scanning electron microscope (JEOL) was used to
obtain the micrographs of both (untreated and
treated) membranes. The mechanical properties of
deproteinated membrane specimen (rectangular,
50×7.5 mm) were evaluated using Goodbrand
Jeffrey Micro 5000 tensile testing machine at a
Table 1
Medium for cellulose production from Acetobacter xylinum
Chemical Concentration (g/l)
Sucrose 50.0
Yeast extract 5.0
Ammonium sulphate 5.0
Potassium hydrogen phosphate 3.0
Magnesium sulphate, hydrated 0.05
 V. Dubey et al. / Separation and Purification Technology 27 (2002) 163–171
cross-head speed of 10 mm/min. An average of
five readings was reported.
2.4. Determination of sorption
The degree of sorption (Q) was determined by
immersing 1.0 g of the membrane in 50 ml of
water –ethanol mixtures at 3091 °C until equili-
bration. The membranes were then removed and
dried between the folds of the filter paper and
weighed using analytical balance (205ACS Pre-
cisa, Switzerland) with an accuracy of 0.1 mg.
From the weight of the dry membrane (wd), the
degree (%) of sorption Q was calculated using the
equation given below:
  n
ws
Q= −1 × 100
wd
where ws is the weight of the swollen membrane.
The increase in weight of the membranes was due
to sorption of water and alcohol.
2.5. E6aluation of solubility selecti6ity
In order to determine the amount of water, in
the membrane phase, the above membrane speci-
mens swollen to equilibrium in binary water– al-
cohol mixtures were desorbed using the
cold-finger technique. The specimen was placed in
a reservoir at 100 °C under a vacuum of 1.0
mmHg for 2 h. The desorbed vapours were col-
lected in a trap cooled by ice– salt mixture at a
temperature of −10 °C. The composition of the
condensate was determined by the measurement
of refractive indices using an Abbe Refractometer
(Model RCR-1, from M/s Rajdhani Scientific In-
struments, New Delhi, India) having an accuracy
of 1×10 − 3 and lower detection limit of 1% for
water –alcohol mixtures. The solubility selectivity
(hs) was calculated using the following equation:
Ym/Xm
hs =
Yf/Xf
where Y and X are the weight fractions of water
and alcohol, respectively, in the membrane (Ym,
Xm) and feed (Yf, Xf).
165
2.6. Per6aporation
The batch-stirred PV cell used for the PV exper-
iments was the same as that of Netke et al. [13].
The effective membrane area was 32.15 cm2. The
membrane was supported by a filter paper over a
porous sintered steel disc 6.4 cm in diameter.
Prior to PV, the specimen was allowed to equili-
brate with the feed solution for 12 h at 30 °C.
After a steady state was attained, the permeate
was condensed in a cold-trap by ice–water mix-
ture at −10 °C. The feed chamber was at 329
1 °C and downstream pressure was maintained at
1.0 mmHg in all the experiments. The flux (J) was
determined by measuring the weight of the perme-
ate. The composition of the feed solution and the
permeate were determined by Abbe Refractome-
(1) ter. The permeation selectivity was calculated us-
ing the following equation:
hp(water/ethanol) =
(Ywater/Yethanol) n (3)
( Xwater/Xethanol)
where X and Y are the weight fractions of species
in the feed and permeate, respectively. The PSI
was calculated from Eq. (4) defined by Feng and
Huang [14].
PSI = J(hp − 1) (4)
3. Results and discussion
A previous study [4] revealed that the produc-
tion of cellulose by Acetobacter is maximised in
sucrose as compared with glucose and fructose.
Also the cellulose mass was higher for all pH
values (4.0–7.0) at 30 °C than that at 20 °C. The
pH variation caused a small alteration (B 5%) in
the amount of cellulose produced. The final pH of
the medium was in the range 2.20–3.24 due to the
evolution of acetic acid [3,4]. For the present
study, the membranes were prepared as per the
conditions stated in Table 1. The thickness of the
membranes was maintained at 100 mm after de-
(2) proteination by NaOH. The membrane was char-
acterised before and after the alkali treatment. All
the membranes used in this study were prepared
in the same batch to ensure identical growth and
thermal history.
166 V. Dubey et al. / Separation and Purification Technology 27 (2002) 163–171
Fig. 1. FTIR spectra of nascent and NaOH treated BCM.
3.1. Characterisation of membrane
The elemental analysis revealed the composi-
tion of carbon and hydrogen conforming to the
structure of cellulose. The amide bond at 1538
cm − 1 in the IR spectra of the untreated mem-
brane disappeared upon exposure to the alkali
(Fig. 1). Low wave number of carbonyl amide
group in untreated specimen is rationalised by
the formation of intermolecular hydrogen bond
between the amide group and an adjacent NH
group. Also the 1650 cm − 1 stretch (CONH)
shifts to 1659 cm − 1 upon alkali treatment as the
CO linkage is freed from NH. The SDS
PAGE electrophoresis reported earlier [4] showed
that the molecular weight of the proteins in the
untreated membrane was in the range 116– 20
kDa. The SEM micrograph (20 000× 10 kV) of
the untreated membrane (Fig. 2a) revealed a
mat-like structure with interwoven microfibrils.
Some intact bacteria were also seen. The fibril
diameter was in the range 0.03–0.05 mm. On
treatment with the alkali, the membrane was de-
proteinated, and a fibrous network resembling a
porous non-woven fabric structure having no
preferential direction of fibres (Fig. 2b) was re-
vealed. These fibres were devoid of any surface
details and associated intact bacteria or debris.
The fibres appeared swollen after the NaOH
treatment, their diameter was in the range of
0.044–0.63 mm. The wide-angle X-ray diffraction
reported by Takai et al. [15] also showed sharp
peaks indicating the crystalline structure of bac-
terial cellulose.
The mesh-like interwoven structure of the
membrane imparts high tensile properties akin to
bonded non-woven fabric. From Table 2, it ob-
served that the tensile strength is of the order of
0.1 N mm − 2 and the Youngs modulus is 102 N
mm − 2. The membrane also possesses excellent
 V. Dubey et al. / Separation and Purification Technology 27 (2002) 163–171 167

Fig. 3. (a) Degree of sorption Q of deproteinated BCM for

water– alcohol binary mixtures; (b) solubility of water in the
membrane phase for water – alcohol binary mixtures.

3.2. Sorption and solubility in the membrane

phase

According to the prevalent solution-diffusion

model, widely used in explaining separation char-
acteristics of membrane materials, the flux and the
separation factor of a component in a binary
system through a membrane are functions of the
solubility and diffusivity of the component. The
solubility of the components in the membrane was
determined using the sorption–desorption
Fig. 2. SEM micrographs of (a) untreated BCM, (b) NaOH method.
treated BCM. The preferential sorption properties of the de-
proteinated membrane were investigated in etha-
chemical resistance. It is insoluble in alcohols, nol –water mixtures of different compositions and
ketones, aldehydes, hydrocarbons, ether as well as the overall degree of sorption was calculated using
aprotic solvents (DMF, DMSO, THF). It is also Eq. (1). The results shown in Fig. 3 indicate that
resistant to dilute acids and bases. It swells in as the water content in the feed mixture increased,
water without dissolution. the degree of sorption Q, also increased from 20
to 135%. The value of Q in pure alcohol was only
18%, it increased to 110% in equi-weight (1:1)
Table 2
Mechanical properties of NaOH treated BCM mixture of water and alcohol and to 135% for
pure water. It indicates that the membrane is
Property Mean value Standard deviation hydrophilic in nature and sorbs almost seven
(S.D.) times more water than alcohol.
Strain @ break (%) 10.50 1.58
In order to determine the relative amounts of
Elongation @ break 3.33 0.54 water and alcohol in the membrane phase sorbed
(mm) from the binary mixtures, the swollen membranes
Stress @ break 0.1 0.03 were desorbed at 100 °C under a vacuum of 1
(N mm−2)
mmHg and the condensate was analysed using
Young’s Modulus 101.6 0.96
(N mm−2) refractive index measurement. From the results
plotted in Fig. 3, it is seen that as the amount of
168 V. Dubey et al. / Separation and Purification Technology 27 (2002) 163–171
Table 3
Sorption selectivity (hs) in NaOH treated BCM for water–ethanol binary mixtures
Feed composition (v/v) Composition (v/v) in the membrane
Water Alcohol Water
90 10 97.5
70 30 93.0
50 50 91.25
30 70 85.0
10 90 30.0
water in the feed increased from 10 to 100%, the
water sorbed in the membrane also increased
from 0.2 to 1.4 g/g. Correlating the above data in
Fig. 3, it can be said that solubility of water in the
membrane phase increases as the overall sorption
increases. This implies that water is sorbed prefer-
entially. The sorption selectivity for water (hs),
calculated using Eq. (2), was in the range of 4– 10.
From Table 3, it is observed that the hs values
were favoured when the feed was richer in ethanol
and contained 30–50% of water by volume. Re-
duced sorption selectivity at higher contents of
water may be attributed to excessive swelling of
macromolecular chains and flow coupling. Due to
interaction with one component viz. water, the
membrane becomes accessible to the other com-
ponent (alcohol, in this case). Hence, the useful
range of sorption is considered to be 5– 25% (refer
Fig. 3); selectivity reduces at higher sorption
values.
3.3. Per6aporation properties
The PV process combines the evaporation of
volatile components of a mixture with their per-
meation through a polymeric membrane under
reduced pressure conditions. It, therefore, involves
a sorption step at the membrane upstream face,
followed by a diffusion through the dense film
and a desorption into the vacuum. Thus PV per-
formance of a membrane, termed PSI is described
in terms of two important parameters namely,
flux (J) i.e. the mass crossing the membrane per
unit area in a unit time and the selectivity towards
the preferentially permeated component. For the
membranes under study, a batch stirred PV cell
hs for water
Alcohol
2.5 4.3
7.0 5.7
8.75 10.4
15.0 13.5
70.0 3.9
was used at a temperature of 309 1 °C. Eqs. (3)
and (4) were used to calculate the permeation
selectivity and PSI values.
3.3.1. Effect of alkali treatment
The cellulose membrane initially used was in
the nascent form without any chemical treatment.
The flux obtained was low (32.3 g m2 h − 1) and so
was the selectivity (4.88) for 1:1 compositions of
alcohol–water mixtures. For the deproteinated
membrane the flux increased considerably to 55.1
g m − 2 h and selectivity was 10.1. The membrane
thickness was about 100 mm. The PV characteris-
tics of the deproteinated membrane were studied
over the entire range of compositions of binary
mixtures.
3.3.2. Effect of concentration
Figs. 4 and 5 depict total flux (J) and selectivity
(hp) as a function of varying feed composition. A
‘trade-off’ relationship between selectivity for wa-
Fig. 4. Water flux of binary water – alcohol mixtures through
deproteinated BCM.
 V. Dubey et al. / Separation and Purification Technology 27 (2002) 163–171
Fig. 5. Permeation selectivity for binary water – alcohol mix-
tures through deproteinated BCM.
ter and its flux is observed from these figures. With
an increase in feed water concentration from 10 to
50%, the flux increases from 112 to 153 g m − 2 h
and water selectivity decreases from 285 to 40 for
all water– alcohol systems. This phenomenon may
be due to plasticizing effect of water, and probable
flow coupling between water and ethanol. As the
water concentration in the feed increases, the
amorphous regions of the membrane swell and the
polymer chains become more flexible, allowing
alcohol molecules also to pass through, thus lower-
ing the ability of the membrane for selective trans-
port. When the concentration of water exceeds 60
wt.%, a reversal in the phenomenon occurs, viz.,
the flux decreased drastically with slight increase in
the selectivity. This may be attributed to the
clustering of water-molecules [7– 11]. The inter-
molecular hydrogen-bonding forms large clusters
of water thereby reducing their diffusivity through
the membrane. Since the swelling of the membrane
is appreciable (Q \ 100%, refer Fig. 3) when the
feed is richer in water, the macromolecular chains
may become flexible due to plasticisation. This
facilitates the solubility of the associated water
molecules in the membrane phase but retards their
diffusivity, and hence the overall flux reduces. This
is supported by the interesting observation that the
flux for pure water through the membrane was
only 31 g m2 h − 1, while the solubility in the
membrane phase was high viz. 1.34 g g − 1. With a
small amount (  10 wt.%) of alcohol in the binary
feed mixture, the overall flux increased to 111 g m2
h − 1. Even though the membrane is hydrophilic,
169
Fig. 6. Pervaporation separation index for binary water – alco-
hol mixtures through deproteinated BCM.
the flux for pure alcohol is substantial (41 g m2
h − 1); while the solubility in the membrane is as low
as 0.19 g g − 1.
The overall contribution of flux and selectivity
to PV is defined by the PSI. The PSI values are
plotted in Fig. 6 as a function of feed composition.
Two distinct regions can be recognised correspond-
ing to water-lean mixtures (B30 wt.%) and water-
rich mixtures. In the former region, PSI is of the
order of 104 g m − 2 h ranging from 13 600 g m − 2
h for 30% water to 32 700 for 10% water in the
feed. For the water-rich region, PSI is lowered and
is in the range 4535–13000 g m − 2 h. It, therefore,
appears that the membrane may be effective in
dehydration of azeotropes of ethanol. This is fur-
ther substantiated by the vapour–liquid equi-
librium (VLE) diagram shown in Fig. 7. The PV
curve for ethanol lies below the VLE, indicating
Fig. 7. Vapour – liquid equilibrium diagram and pervaporation
curve for binary water – alcohol mixtures.
170 V. Dubey et al. / Separation and Purification Technology 27 (2002) 163–171

Table 4 Mumbai, for fruitful discussions. We thank Dr

Flux and selectivity of ethanol–water (70:30 wt.%) through
homogeneous membranes (50 mm) at 70 °C (ref. [16])
R.V. Swamy, Chief Controller (R&D), DRDO,
New Delhi for keen interest and invaluable sug-
Polymer Flux (kg m2 hp gestions. Dr D.K. Jaiswal, Director, DRDE,
h−1) Gwalior is thanked for providing the necessary
facilities in the laboratory. Our thanks to T.K.
Poly(acrylonitrile) 0.007 12 500
Das for secretarial assistance.
Poly(acrylamide) 0.011 4080
Poly(vinyl alcohol) 98% 0.08 350
Poly(ether sulphone) 0.072 52
Polyhydrazide 0.132 19
Bacterial cellulose membrane, 0.112 287
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The PV characteristics of deproteinated BCM
were investigated over the wide range of water–
ethanol feed composition. The membrane is
promising for dehydration of azeotropes of etha-
nol. It has a high selectivity towards water and a
reasonable flux.
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