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

Producing Ethanol from Starch

SATYA N ARAYANA MANDAVILLI

Department of Chemical Engineering, University of Puerto Rico,

Mayaguez, PR 00681-9046

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)

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

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 )

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)

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

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)

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

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

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

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

concentration as a function of position in the reactor,

888

Fig. 4

= C1 / t

(5)

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

(6 )

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)

(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)

(11)

Fig. 5

enzymatic kinetics

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.

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

inhibition model for different initial substrate concentration

Fig. 7

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

=

=

=

=

=

=

=

=

=

=

=

=

=

=

=

=

=

=

=

=

=

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)

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