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Research Paper

Journal of Chemical Engineering of Japan, Vol. 33, No. 6, pp. 886890, 2000

Performance Characteristics of an Immobilized Enzyme Reactor


Producing Ethanol from Starch
SATYA N ARAYANA MANDAVILLI
Department of Chemical Engineering, University of Puerto Rico,
Mayaguez, PR 00681-9046

Keywords: Bioreactor, Immobilized Enzyme, Mass Transfer, Reaction Kinetics, Modeling


The utility of an immobilized enzyme, gluco-amylase on cellulose suppor t, for the hydrolysis of potato
starch, has been studied in a fixed bed biochemical r eactor to evaluate the influence of flow parameters
on conversion. The effect of parameters like pellet size, packed bed height and substrate concentration,
has been studied. Maximum conversions ar e obtained for pellet size of 0.57 cm, packed bed height of 70
cm, and substrate concentration of 10 mg/ml. The kinetic behavior of heterogeneous enzyme, has been
studied, with the aid of an appr opriate mathematical model, which takes into account, simultaneous
mass transfer and reaction kinetics. Based on the experimental observations and the mathematical model,
intrinsic kinetic parameters ar e determined by decoupling dif fusion effects. This knowledge is used in
the design of an immobilized enzyme r eactor.

Introduction
Heterogeneous enzymatic catalysis includes:
a. mass transfer of substrate to the external surface of
catalyst pellet,
b. diffusion of substrate from the external surface to
domain of enzyme,
c. adsorption of substrate,
d. enzymatic reaction,
e. desorption of product,
f. diffusion of product back to the surface of catalyst,
and
g. mass transfer of product to the bulk of fluid stream.
Steps (a) and (g) are external, while (b) and (f) are
internal mass transfer effects. These are essentially
different from homogeneous kinetics. The overall rate
of an immobilized enzymatic process is always influenced by these mass transfer effects. These effects may
become even rate limiting steps.
The rate of external mass transfer of component
A, is given by
NA = KLa(Cb Cs)

(1)

where KL is the external mass transfer coefficient, for


an external area, a, of catalyst pellet. Cb and Cs are
concentrations of diffusing component in the bulk solution and on the catalyst surface, respectively.
For the range of conditions normally encountered
with the immobilized enzymes, Wilson and Geankoplis
(1966) proposed suitable equations for external mass
Received on January 6, 2000. Correspondence concerning this
article should be addressed to S. N. Mandavilli.

886

transfer in terms of J D factors.


Correlations for predicting dispersion coefficients
in a fixed bed are presented by Wen and Fan (1975).
The significance of axial dispersion depends upon the
length of the reactor, the effective diffusivity and the
velocity. Its importance decreases as length and velocity increases. Smith (1981) reported that for low velocity (NRe < 1) and short reactors, axial dispersion
cannot be neglected. The mechanism of internal mass
transfer in liquid filled pores is generally represented
by pore surface diffusion and pore volume diffusion.
Molecular diffusion resulting from differences in concentration between regions of a mixture is expressed
by
J A = CDABX a / Z

(2 )

where JA * is the diffusion flux, for a concentration


change of a diffusing component, X a, as a function of
diffusion distance, Z. D AB is the diffusivity of A in B.
The effective pore volume diffusion coefficient,
Deff, greatly depends upon the pore size and structure
of the particle. When the pore radius is much greater
than the radius of the solute, the particle-wall interactions can be neglected. The effective diffusion coefficient can then be, as reported by Satterfield (1970):
Deff = p DAB / T

(3)

where p is the void fraction of the pellet.


Accurate values of tortuosity factor, , are to be
determined experimentally, though considerable
number of theoretical predictions are available in litCopyright 2000 The Society of Chemical Engineers, Japan

Fig. 1

Schematic representation of activation of cellulose


using 2,4,6-trichloro-s-triazine and covalent linkage enzyme to support

erature. Effective diffusivity measurements of a solute within a porous particle, are made using a chromatographic method involving the response to a pulse
input of solute to a column packed with the porous
particles of interest.
The effect of mass transfer on reaction rate, is indicated by the effectiveness factor, , which is a function of Thiele modulus, . in turn is a function of
characteristic length of the catalyst pellet, L. Thiele
modulus is given by Wadiak and Carbonell (1975), as:

= L[r(Cs)/Deff Cs]1/2

(4)

r, is the distance in radial direction of pellet and Cs, is


the concentration on the pellet surface.
The purpose of the present study is to characterize the free enzyme and experimentally observe the
changes in measured parameters upon immobilization.
The program includes study of both external and internal mass transfer effects in a fixed bed biochemical
reactor packed with cellulose pellets with immobilized
enzyme, glucoamylase. The reaction taken up for study
is, hydrolysis of potato starch to glucose. The experiments are conducted over a range of experimental conditions. The physical properties such as external mass
transfer and dispersion coefficients are estimated from
available correlations. The effective diffusivity is determined experimentally. A suitable mathematical
model is developed to account for the mass transfer
effects and the non-ideality of the reactor.
1.

Experimental

1.1 Preparation of starch solution


Starch solution is prepared by placing a desired
amount of starch in a volumetric flask and made into a
slurry with a small quantity of acetate buffer of pH
7.0. Fresh sodium acetate buffer solution of 0.05M,
4.1 g/l, pH 4.8 is prepared in double distilled water
thrice a week. One percent bacterial -amylase is then
added to this slurry and the temperature is raised to

Fig. 2

Effect of bed height on conversion

80C and held there for one hour. The pH of the solution is checked to ensure that it is at 7.0, otherwise
adjusted using dilute acetic acid.
1.2 Preparation of enzyme in buffer solution
A solution of glucoamylase is prepared by taking
a weighed quantity in the sodium acetate buffer. The
solid insoluble material is filtered. The filtrate contains
the protein that catalyses the reaction, starch to glucose. The protein content in the enzyme is determined
by Lowrys method as reported by Anderson and
Morrison (1925). It is found that 1000 mg of enzyme
contains 63.7 mg of protein. All calculations in this
work are based on the amount of pure glucoamylase
taken for conversion.
1.3 Method of immobilization and optimization of
parameters
Cyanuric chloride reacts rapidly with the free hydroxyl groups present in cellulose. The dichloro-striazyl cellulose may then be reacted with the enzyme
and finally the third chloro group substituted with
amine or other chemical group. A schematic representation is given in Fig. 1. 0.5 g of 2,4,6-trichloro-s-triazine is dissolved in 50 cc of 1:1 acetone water mixture at a temperature of 40C. To this is added 10 g of
cellulose pellets and reacted under stirring for 2 minutes. A mixture of 12 cc of 1.0 mol/liter hydrochloric
acd and 8 cc of sodium carbonate solution is added
and stirred for further 5 minutes. The reaction mixture
is filtered and the cellulose-di-chloro-s-triazyl obtained
is washed first with distilled water and then with acetate buffer. The activated pellets are suspended in
glucoamylase solution. The mixture is incubated at 4C
for 24 hours. The enzyme-cellulose complex is then
washed with acetate buffer until no protein is detectable in the washings. The effects of time, temperature
and pH of activation are studied to get the optimum
values as, 4 minutes, 40C and pH of 5.6. The results
indicate that the enzyme requires about 24 hours of
incubation time to completely saturate all the active
sites on the activated cellulose complex.
887

Fig. 3

Effect of pellet size on conversion

1.4 Fixed bed biochemical reactor


The material of construction for the reactor and
substrate storage tank are entirely glass. Small pieces
of teflon, are used to connect the glass tubing.
The fixed bed biochemical reactor is a straight
long tube wrapped with heating tape. Heat input to the
reactor is regulated by a variac. The neck at the bottom of the column functions as a feed inlet. A stainless
steel plate fixed at the base of the reactor, supports the
packings in the column. The projections along the
length of the column serve as product outlets at various bed heights. The bottom of the column just below
the packing support is v-shaped to eliminate entrance
effects.
The reactor is 105 cm long and has an internal
diameter of 4.51 cm. Constant temperature feed solution is passed through the column. The flow rate is set
to the desired value by adjusting the length of the tube
in the Mariots bottle and by manipulating the glass
valves. The temperatures in the column are monitored
at the inlet, middle and just above the packing with the
help of iron-constantan thermocouples. When the system reaches steady state, samples are withdrawn and
analysed.
1.5 Assay methods
Protein assay is performed by Lowrys method.
Starch and glucose are estimated by a spectrophotometric method.
1.6 Effects of bed height, pellet size and substrate
concentration
Experiments are conducted to study the effects of
bed height (corresponding to various packed weights
of catalyst), pellet size and substrate concentration, on
conversion of glucoamylase treated starch. The results
are presented in Figs. 2, 3 and 4.
2.

Modeling

A differential equation, expressing the substrate


concentration as a function of position in the reactor,
888

Fig. 4

Effect of substrate concentration on conversion

is derived from material balances, as

V0C1 / Z EL 2 C1 / Z 2 + 3 / R(1 ) K L C1 C1, R


= C1 / t

(5)

where V0 is superficial average velocity, C1 and C1,R


are substrate concentrations in liquid phase and at outer
surface of catalyst respectively, and R is the distance
from reactor tube center.
Inside the pellet (solid phase) a mass balance for
the substrate can be made on a spherical shell of thickness, r. Dividing throughout by 4r2r and letting r
0, we get

( )) = pC1, r / t

Deff / r r 2C1, r / r p R C1, r

(6 )

where C1,r is the substrate concentration in the solid


phase of catalyst at distance, r, and p and p are pellet
density and porosity.
The boundary and initial conditions are

C1,r/r = 0 at r = 0

(7)

Deff(C1,r/ r) = KL(C1,r C1) at r = R

(8)

C1,r = 0 at t = 0, 0 r R

(9)

By introducing the fractional conversion, X and suitable dimensionless groups, equations can be written
in the dimensionless form, for steady state.
(1/Pe)(2X1/Y2 X1/ Y) + U(X1, 1 X 1) = 0

(10)

where is Pe is Peclet Number, Y is dimensionless diffusion length, U is dimensionless velocity.

2X1,/ 2 + (2/ )(X1, / ) + 2R(X 1,) = 0

(11)

JOURNAL OF CHEMICAL ENGINEERING OF JAPAN

Fig. 5

Comparison of effectiveness factors for different


enzymatic kinetics

X1, and X1 are dimensionless substrate concentrations.


The Gauss-Siedel over relaxation technique is
employed to numerically integrate Eqs. (10) and (11).
The resulting partial differential equations are converted to finite difference equations. Substituting the
boundary conditions, these equations are solved by a
computer program. The diffusion and reaction model
equations (Eqs. (5), (10) and (11)) include the effect
of external mass transfer, internal mass transfer and
axial dispersion. The accuracy of the model greatly
depends on the proper estimation of the physical constants. Toda and Shoda (1975) measured the effective
diffusivity of a substrate into the plain carriers in the
absence of immobilized enzyme. Bollmeier and
Middleman (1979) inactivated the immobilized enzyme
carrier before measuring the effective diffusivity. The
same procedure was adopted by Sirroti and Emery
(1983) for measurement of the effective diffusivity of
glucose, cellobiose into an immobilized enzyme carrier. In the present work, the tortuosity factor is determined by the conventional manner from = D/Deff,
where is pellet void fraction. It is found to be 1.49,
confirming the literature value for pelleted catalysts.
A similar procedure is adopted for modeling the effectiveness factor. The effectiveness factor, becomes a
function of six parameters, K1, K2, K3, X1, B1, . K1
= KM/S0; K2 = KI/S0; K3 = KP/S0. is calculated with no
external mass transfer resistance when B1 = .
3.

Results and Discussion

Predictions of final conversions of a fixed bed


biochemical reactor, are calculated by solving the diffusion and reaction model. The best set of intrinsic kinetic constants are evaluated for immobilized glucoamylase.
The kinetic constants predicted well all the data
including the reaction carried out by using different
starch concentrations. Compared with homogeneous
kinetics, V max, KM and KI of heterogeneous kinetics decreased. By using the heterogeneous glucoamylase kiVOL. 33 NO. 6 2000

Fig. 6

Comparison of effectiveness factors for substrate


inhibition model for different initial substrate concentration

Fig. 7

Comparison of effectiveness factors for substrate


inhibition model for different amounts of conversion

netic constants and assuming no external mass transfer resistance, theoretical effectiveness charts are prepared for various kinetic models.
Comparison of effectiveness factors for different
kinetic models is given in Fig. 5. The substrate inhibition model always experiences higher effectiveness
factors than other models. Under certain circumstances,
the effectiveness factor may even exceed unity. Because the enzyme molecules are protected by a diffusion barrier, substrate plus product inhibition offsets
the effect of diffusion resistance on substrate inhibition and in turn gives a lower effectiveness factor. The
product inhibition model depletes the effectiveness
factor further.
The effect of initial substrate concentration on
effectiveness factor and Thiele modulus curves for
substrate inhibition model are plotted. A comparison
for various initial substrate concentrations, is given in
Fig. 6. Higher substrate concentrations give higher
effectiveness factors. The curves approach first order
limit at low substrate concentration and zero order limit
at high substrate concentration.
Figure 7 simulates the real reaction environment
of a fixed bed reactor. For a given substrate concentra889

tion and Thiele modulus, and enzyme catalyst with inhibition kinetics, the highest effectiveness factor is at
the entrance of the reactor. The effectiveness factor
drops along the reactor due to decrease of substrate
concentration and increase of product concentration.
Conclusion
The reaction velocity is proportional to the enzyme
concentration in both the cases of free and immobilized enzyme.
The homogeneous kinetics of glucoamylase is best
represented by a modified substrate inhibition model
incorporated with an adjustable parameter. The equation is, V = Vmax S(A + BS)/(KM + S + 2/KI). The kinetic
constants are V max = 6.54 mol/min/mg.protein, KM =
6.57 mg/ml, KI = 310.34 mg/ml, A = 5.18 and B =
0.19 ml/mg. The temperature stability of immobilized
glucoamylase is superior to that of soluble enzyme.
Free glucoamylase lost all its activity at 70C upon
incubation for one hour whereas immobilized enzyme
retained 83% of initial activity.
Immobilized enzyme is stable in lower pH ranges.
Enzyme progressively gets denatured as pH is raised.
The diffusion and reaction model which includes
external and internal mass transfer effects and axial
dispersion predicts quite well the performance of the
fixed bed reactor. By solving the simultaneous diffusion and reaction model numerically, best kinetic constants are obtained as Vmax = 5.3 mol/min/mg.protein,
KM = 4.47 mg/ml, KI = 211.03 mg/ml, A = 5.18 and B
= 0.19 ml/mg.
The theoretical analysis of effectiveness factor in
this study proved useful for judging the significance
of diffusion resistance in the overall rate process of
biological systems.
Nomenclature
A
= cross-sectional area of fixed bed reactor
[cm 2]
a
= external area of catalyst pellet per unit weigh of
mass
[cm 2/g]
B1
= Biot number K LR/D eff
Cb
= concentration of diffusing component in bulk solution
[mmol]
C0
= initial substrate concentration at entrance of fixed
bed reactor
[mmol]
C1
= substrate concentration in liquid phase in fixed bed
reactor
[mmol]

890

C 1,r

D AB
D eff
Dp
EL
J A*
KI
KL
KP
KS
L
NA
Pe
R
r
T
t
V0
X1
X1,
Y
Z

=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=

substrate concentration in solid phase of catalyst


at distance, r
[mmol]
molecular diffusivity of diffusing solute [cm 2/s]
effective diffusivity
[cm 2/s]
effective diffusivity of product
[cm 2/s]
axial dispersion coefficient
[cm 2]
diffusion flux
[mol/cm 2/s]
enzymatic substrate inhibition
[mg/ml]
external mass transfer coef ficient
[cm/s]
mass transfer coefficient for product
[cm/s]
mass transfer coefficient for substrate
[cm/s]
characteristic length
[cm]
rate of external mass transfer
[g/s]
Peclet number LV0/ EL
pellet radius
[cm]
distance in radial direction of pellet
[cm]
absolute temperature
[K]
time
[s]
superficial velocity
[cm/s]
(C0 C1 )/C0
(C0 C1,r)/C0
Z/L
distance in the direction of dif fusion
[cm]

=
=
=
=
=
=
=

D S/D
bed void fraction
void fraction of pellet
r/R
pellet density
tortuosity factor
Thiele modulus

Literature Cited
Anderson, T. W. and K. D. Morrison; Estimation of Protein by
Lowrys Method, J. Biol. Chem ., LXV, 393398 (1925)
Bollmeier, J. P. and S. Middleman; Inactive Immobilized Enzyme
Carrier for Measurement of Ef fective Diffusivity, Biotech.
Bioeng., 21, 23032307 (1979)
Satterfield, C. N.; Mass Transfer in Heterogeneous Catalysis, MIT
Press, Cambridge, USA (1970)
Sirotti, D. A. and A. Emery; Diffusivity of Glucose and Cellobiose into an Immobilized Enzyme Carrier , Biotech. Bioeng.,
25, 17731777 (1983)
Smith, J. M.; Chemical Engineering Kinetics, 3rd ed., McGraw Hill,
New York, USA (1981)
Toda, K. and M. Shoda; Measurement of Ef fective Diffusivity of
Substrate in the Absence of Immobilized Enzyme, Biotech.
Bioeng., 17, 481485 (1975)
Wadiak, D. T. and R. C. Carbonell; Ef fect of Mass Transfer on
Reaction Rate, Biotech. Bioeng., 17, 176182 (1975)
Wen, C. Y. and L. T. Fan; Models for Flow Systems in Chemical
Reactors, Mercel Dekker, New York, USA (1975)
Wilson, E. J. and C. J. Geankopolis; J D Factor for External Mass
Transfer in Heterogeneous Chemical Reactors, Ind. Eng.
Chem., 5, 916 (1966)