Indian Journal of Chemical Technology
V()12.July Il}l}:\pp. 179-IRJ
Enzymatic hydrolysis of sucrose in a dead-end stirred cell membrane
bio-reactor using asymmetric cellulose acetate membranes
Vineet Kumar", N,Ravi Kumar" & S N Upadhyay'"
~Department of Chemical Engineering & Technology, "School of Biochemical Engineering, Institute of Technology,
Banaras Hindu University, Varanasi 221 00)" India .'
( Asymmetric cellulose acetate membranes have been prepared and characterized
with a view to use them in membrane bio-reactors. Enzymatic hydrolysis of sucrose
gated in a dead-end stirred cell type membrane bio-reactor using these membranes.
ent operating variables such as substrate concentration, enzyme loading, flow rate
lume have been studied.)
") ./: "{.J)
Membrane reactor is one of the first novel enzyme re~~t~;' systems developed in the sixties. It
consists of semipermeable membrane made of
cellulose acetate or other polymeric materials
having tailored molecular cut-off characteristics
placed at the bottom of the reactor. Ghose and
Kostic! used <Lead-end S~~!!~<!..c~lI(DESC) membrane bio-reactor for continuous enzymatic saccharification of cellulose by cellulase. Butterworth
et al? demonstrated the feasibility of using an ultrafiltration membrane reactor for the continuous
hydrolysis of starch by a-amylase, and by glucoamylase. Swanson et al.' used DESC reactor
system for the hydrolysis of starch using amylases.
Enzymatic hydrolysis of sucrose in a thin channel
ultrafiltration system has been studied earlier">. A
mathematical model was developed to simulate
the complete transient and steady state behaviours of the experimental reactor. These reactors
have been used by several researchers6-!5 either
for enzymatic conversions or in fermentation processes, in view of their numerous advantages compared to other type of conventional bio-reactors.
Membrane bio-reactors, whether used for enzymatic or microbial conversion processes, provide
an opportunity to vastly improve the process efficiency and productivity. One of the major advantages of these reactors is the possibility of rapid
removal of inhibitory or toxic metabolic products
of lower molecular weight. High productivity (kg
m ? h-1) has been reported in such systems14-20.
They also permit the use of high bio-catalyst concentrations and high flow rates (dilution rate). In
enzyme catalysed reactions, the enzyme can be
recovered and/or reused, thus enzyme utilization
is increased and production costs are lowered.
* Author to whom correspondence
should be addressed.
in the laboratory
has been investiEffect of differand working vo-
product stream from membrane bio-reactor is
free from microbial cells or particulates. This
helps in reducing the downstream processing
costs. In the present work, cellulose acetate membranes are prepared, characterized and used in
the membrane bio-reactors for investigating the
enzymatic hydrolysis of sucrose.
Experimental Procedure
Chemicals-LR
grade cellulose acetate (BDH)
was used for making membranes. LR grade acetone (Quligen Fine Chemicals) was used as solvent and LR grade formamide (BDH) and AR
grade magnesium perchlorate (CDH) were use~
as swelling agents. LR gr~de urea. (Sarabhai
Chemical Co.), sucrose (s.d, Fme Chemlca~s), polyethylene glycol MW 4000 (CDH), pol~myl pyrrolidone MW 30000 (CDH) and polyvinyl alcohol MW 125000 (s.d. Fine Chemicals) were used
as the solutes for permeation tests. Distilled water
was used as solvent in all the experiments.
Substrate and enzyme-The
substrate chosen
"was sucrose (s.d, Fine Chemicals). The substrate
solution was taken in a feed tank and it was fed
to the reactor with the help of compressed air
supply connected to the tank.
.
The enzyme selected in this study was invertase. Commercial Baker's yeast (Saccharomyces
cerevesiae) cells (cake form) were used as the
source of invertase enzyme. The required amount
of enzyme (as weighed cells) disp~rsed in a sI?all
amount of substrate solution was introduced mto
the reactor with the help of a syringe. The enzyme levels are expressed in mg cells/mL.
Membranes- The cellulose acetate (CA) membranes used in the reactor were prepared in the
laboratory by solution casting method, Chemically
pure cellulose acetate powder was dissolved in
180
INDIAN J. CHEM. TECHNOL., JULY 1995
acetone and required amount of swelling agent
(formamide or magnesium perchlorate) was added
to the solution. The membranes of 0.006 to 0.01
ern thickness were cast and characterized for pore
size, porosity, pore density, water flux and rejection of various solutes 15. Based on these studies
membrane prepared from a solution of 20 g
CA+ 88.5 mL acetone + 20 mL magnesium perchlorate in distilled water (10% w/w) was used in
the DESC. This membrane had a porosity of
0.5374, average pore size 0.045 ,urn and pore
density 33 x 107 cm-2. It was thus suitable for retaining molecules larger than 4000 MW.
Dead-end stirred cell (DESC) membrane reactor
system- The reactor consisted of two chambers (i)
feed chamber (upper chamber) and (ii) product
chamber (lower chamber) separated by a membrane. The DESC assembly is shown in Fig. 1.
. The reactor was provided with an inlet having a
needle valve for regulating the feed entering the
chamber. It was also ..provided with a magnetic
stirrer, and a pressure gauge. The permeate containing the product get collected in the product
chamber and came out through the outlet.
The enzyme (bounded inside cells) was retained
in the reactor as it could not come out through
the membrane. Only product and sucrose could
pass through it. Thus.: enzyme was continuously
reused without any loss. All measurements were
made at a temperature of 20-22°C and pressure
applied was varied from 0.34 to 1.4 x 105 Pa to
maintain appropriate permeation rate across the
membrane. Amount of reducing sugar present in
the permeate was monitored spectrophotometrically using DNS method" and was used to follow
the reaction rate.
Storage
Vessel
ognetic Sturrer
Pressure Gouge
Permeate
Compressor
Experimental
set-up
Fig. l-e-Schematic diagram of the experimental set-up
Results and Discussion
Membrane characteristics-Most
important properties of a membrane are pore size, pore density
(number of pores per unit area), porosity (void
fraction) and solvent and solute fluxes. Average
pore diameter of thernembrane were determined
by the method proposed by Blumberg". The
pores are assumed to be cylindrical capillaries of
uniform length and diameter and Hagen- Poiseuille
equation is applicable for flow through pores.
Thus the permeation velocity, u (em s -1) is given
by
u=ste dt, I'1PI(32om,u)
... (1)
where e is the porosity, dp is the pore diameter,
I1.P is trans-membrane pressure difference,
is
the membrane thickness and ,u is the viscosity of
the liquid. Permeation velocity of water at a particular pressure was determined experimentally and
d was calculated with the help of above equation.
P~rosity of the membrane used in Eq. (1) was determined by a very simple technique, in which a
piece of wet (surface dried) and completely dried
membrane of known dimensions (in wet condition) was weighed. The difference in two weights .
were used to determine the volume of water
trapped in pores. The ratio of pore volume to the
total volume of the membrane (in wet condition)
gave the porosity of the membrane. Knowing pore
diameter and porosity, pore density (number of
pores, em - 2) were calculated by the following
equation,
om
4eom
N=--,
...
(2)
jfd~
Permeability tests of the membrane were carried out with aqueous solutions of different solutes
(urea, MW 62; sucrose, MW 342; polyethylene
glycol (PEG), MW 4,000; polyvinyl pyrrolidone
(PYP K30), MW 44,000; polyvinyl alcohol (PYA),
MW 120,000) in the trans-membrane pressure
difference range of 0-1.4 x 105 Pa. Curves obtained for permeate flux and % rejection are
shown in Figs 2 and 3, respectively. These figures
exhibit the typical behaviour of concentration polarisation and rejection with increasing transmembrane pressure.
Typical effect of membrane thickness on pore
size of membranes cast using formamide and
magnesium perchlorate is shown in Fig. 4. It is
seen that for a given composition of swelling
agent (forrnarnide or magnesium perchlorate) and
solvent (acetone), the pore size increases with in-
181
KUMAR et af.: ASYMETRIC CELLULOSE ACETATE MEMBRANES
!
>u•
e
'<If
0'020
0·010
0,005
0.020
0'0000'0
,
,,,
/
0.018
0·015
,
/
E 0.016
/
:>.
,
/
/
,"
I!I
.;
.~ 0.014
~
o
•
0'4
0'2
,
0'6
,
0'8
,PYA
"0
PEG 4,000
a. 0.012
pyp K 30
0.010
- 8- •••••••
110,000
1·2
1'4
0.008
0.003
Trons-m.mbran. pr.ssur. dilf.r.nc., (Pol x I<f"S
Fig. 2-Variation
of permeation velocity with trans-membrane pressure drop for aqueous solution of urea (9.125
glmL), sucrose (9.75 glmL), PEG (9.375 glmL), PVP (3.6
glmL) and PYA (3.2 glmL)
0.004
Fig. 4-Effect
100
-
"
um ••••••
Fonnamide
~
-
.Q -
0.005
0.006
0.007
0.008
Membrane thickness. em
0.009
_.1
0.010
of membrane thickness on pore density
-
--<l- - -
90
PYA120,000
II a.0uc
•c:•
~ ~
Of
C
80
70
60
~
20
o
0-0
0'2
0'4
Trans-m.mbran.
0'6 -
0,8
NY
prHsur. dill••• nc•• (Pal"
1·2
"'4
10-5
Fig. 3-Variation
of % rejection with trans-membrane pressure drop for aqueous solution of urea (9.125 glmL), sucrose
(9.75 glmL), PEG (9.375 g/mL), PVP (3.6 g/mL) and PYA
(3.2g/mL)
creasing thickness. This increase is much steeper
for perchlorate based membranes than for formamide based. The porosity and pore density both
decrease with increasing membrane thickness.
Effect of swelling agent on porosity, pore size
and pore density has also been investigated. It has
been observed that as the amount of swelling
agent is increased the porosity and pore size also
increase. This increase is steeper in the case of
magnesium perchlorate based membranes compared to formamide based membranes. on pore
density, the swelling agent has effect opposite to
that on porosity and pore size. Further, beyond a
certain amount of formamide there appears to be
little change in these parameters.
Enzymatic hydrolysis of sucrose-Effects
of different operating variables such as substrate con-
o
2
3
4
Substrate. mg/mL
5
6
7
Fig. 5-Effect of substrate concentration on per cent conversion (£=0.3 mglmL; J=5 mUmin; V= 1,000 mL; pH=6.8
and T=21 ± 1°e)
centration, enzyme loading, flux and working volume on per cent conversion have been studied.
Fig. 5 shows the effect of substrate concentration
on per cent conversion. At 1, 2 and 3 g L- 1 substrate (sucrose) concentrations,
approximately
95% conversion is observed which is nearly the
maximum or the saturation value. After that, as
the substrate concentration increase, the percentage conversion gets reduceq. It drops down to
54% for 6 g L -1 compared to 95% for 3 g L-.l
sucrose concentration.
Fig. 6 shows the effect of enzyme (cell) loading
on per cent conversion. A rapid initial increase in
the percentage conversion at low enzyme loading
is observed. As the enzyme loading is increased,
the rate of increase in the percentage conversion
is found to decrease.
Fig. 7 shows the effect of permeate rate on percentage conversion. As the permeation rate is increased, percentage conversion is found to de-
182
INDIAN 1. CHEM.TECHNOL.,
JULY 1995
100
co
80
co .60
'iii
...
'iii
•....
~
c
Q)
>
60
c
oo
-
840
-
C
C
Gl
~
lI>
o
•....
cf 20
lI>
Q.
20
o
o
0.0
o
0.1
0.2
0.3
0.4
Enzyme loading, mg/mL
0.5
0.6
Fig. 6-Etlect of eniyme loading .on per cent conversion
(5=5.0 mglmL; J=5 mUmin; V=l,OOO mL; pH=6.8 and
T= 21 ± 1°C)
100
2l.l0
400
tiOO
800
1000
1200
Working volume, mL
Fig. 8-Effect of working volume on per cent conversion
(5=5:0 mglmL; £=0.3 mglmL; J=5 mUmin, pH 6.8 and
T=2-1 ± 1°C)
sumptions of this model are: m irreversible conversion of substrate to product with no inhibition,
(ii) a kinetically controlled process, (ill) an ideal
90
mixed CSTR system, (iv) applicability of first or"
o
der rate expression with respect to effective sub'iij
~
80
strate
concentration in the CSTR, (v) negligible
>
"o
mass transfer limitation and adsorption of enzyme
o
on the membrane and (vi) negligible hydrolysis of
~ 70
~
sucrose without enzymatic activity.
Q.
Upon entering the reactor the substrate So
60
present in the feed reacts with the enzyme E,
forming a product P. There will be some amount
of unconverted substrate S in the reactor all the
50
2
3
4
5
7 time. The level of S will depend upon the .activPermeote rote. mLftnin
ity of enzyme and membrane pore size. Further
for
an ideal well mixed CSTR, substrate concenFig. 7-Effect of permeate flux on per cent conversion
tration
in the reactor and outlet are the same.
(5=5.0 mglmL; £=0.3 mglmL; V=l,OOO mL; pH=6.8 and
T=2l ± 1°C)
Hence, the rate of reaction is proportional to the
substrate concentration S. The product P permeating out of the reactor consists of the procrease in a non-linear fashion. At low flow rates,
the residence time for the substrate molecules will ducts formed during the reaction. The remaining
components of the reaction mixture are retained
be high and hence a higher percentage conversion.
in the reaction vessel together with some product
Fig. 8 shows the effect of working volume on molecules due to equilibrium partitioning nature
of ultrafiltration membrane.
per cent conversion. A~ higher volumes, converThe fractional conversion X of substrate to the
sion is found to be high. This is due to the fu~
creased residence time of substrate molecules at product can be written as
higher workipg volumes .
... (3)
X=P/So
Attempts were also made to observe the adFrom material balance
sorption, if any, of enzyme or substrate on reactor
walls and membrane, and practically no detectS=So-P=So(1-X)
... (4)
able adsorption was observed.
Assuming the validity of Michaelis-Menten kinetic
Kinetic modelling of the process-For develop- model the rate of reaction, r, can be written as
ing a kinetic model for the substrate-enzyme system used in this work, approach used by Deeslie
... (5)
and Cheryan23 has been followed. The major as-'
lD
"
I'
"I
183
KUMAR et al.: ASYMETRIC CELLULOSE ACETATE MEMBRANES
c0u>
1.0
KmX
:8
'fcu 0.60
"60.4
c
3'~0.2
X+ '1- X) So-
0.80.0
Space time.
t
X +._m ___
10
I
S.(1-X)
,JO
20
40
50
K t
K
,
K2l'
•••
(10)
In order to evaluate the performance of the
reactor used in this work, experimentally determined fractional conversions are plotted against l'
in Fig. 9. Model predictions are also shown by a
continuous line. It is seen that the measured conversions are higher than the prediction at low
space times and a good correlation is observed at
higher space times.
X
2
References
Fig. 9-Performance
curves for membrane reactor system
(line is predicted curve based on kinetic model and point values are experimental data for 5 mglmL of sucrose)
where Km is the Michaelis-Menten constant and
Vmax is the maximum reaction rate given by
... (6)
Vmax=K2E
where K 2 is the specific reaction rate and E is the
totaleilzyme
concentration in the CSTR. From
mass balance one can write
J. p= r. V
... (7)
where J is flow rate of feed which is equal to
permeate rate and V is the reaction volume. From
Eq. (4) through Eq. (7) one may write
KmX
XSo+
I.
u,
EV
=K2
J
=K2E¢>
... (8)
where = VI J is the residence time.
Eq. (8) is similar to the relation used by other
workers for describing the kinetics of an ideal
continuous flow reactor, where (VI J) is expressed
as the fresh feed residence time. Deeslie and
Cheryan23 suggested the use of a modified residence time ( l') rather than ¢>, where l'is defined as
¢>
l'=(EVIJSo)
...
(9)
The advantage of l' over
is that all variables
affecting the reactor are grouped conveniently into one term. The units are still in terms of time.
Thus one can write
¢>
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