Summary Glucose oxidase containing catalase was immobilized with a copolymer of phenylenediamine and glutaraldehyde on pumice and titania carrier to study the enzymatic oxidation of glucose in a differential-bed loop reactor. The reaction rate was found to be first order with respect to the concentration of limiting oxygen substrate, suggesting a strong external mass-transfer resistance for all the flow rates used. The partial pressure of oxygen was varied from 21.3 up to 202.6 kPa. The use of a differential-bed loop reactor for the determination of the active enzyme concentration in the catalyst with negligible internal pore diffusion resistance is shown. Catalyst deactivation was studied, especially with respect to the presence of catalase. It is believed that the hydrogen peroxide formed in the oxidation reaction deactivates catalase first: if an excess of catalase is present. the deactivation of glucose oxidase remains small. The mathematical model subsequently developed adequately describes the experimental results.
INTRODUCTION Immobilized enzyme reaction systems exhibit many of the same characteristics as conventional heterogeneous catalytic reactions. Problems of transfer to the catalyst exterior and porous diffusion of products and reactants through the interior of the catalyst support are very similar although sometimes the larger molecular size of biological compounds must be considered. Catalyst deactivation is probably much more important for the relatively unstable enzymes than for conventional heterogeneous catalysis. Optimal conditions of temperature and pH exist for all enzyme systems. Many of the experimental techniques developed for heterogeneous catalysis can be employed in enzyme systems. Of course, the basic approach to chemical kinetic and overall reactor modeling remains the same. Biotechnology and Bioengineering, VOI. XXI, Pp. 89-109 ( 1979) 0006-35921701OO2 I -0089$01 .OO @ 1979 John Wiley gi Sons, Inc.
90
PRENOSIL
The system described here was chosen for its suitability as an academic problem in enzyme reactor engineering. As such it possesses most of the complexities that would possibly be found in a heterogeneous catalytic reaction. The reaction system is as shown: glucose lactone
+ 0,
+ H,O
GI0
lactone
+ HzOZ
gluconic acid
(1)
Glucose is oxidized to gluconolactone and hydrogen peroxide by glucose oxidase (GIO). Catalase (Cat) catalyzes the decomposition of hydrogen peroxide to oxygen and water. The gluconolactone undergoes rapid hydrolysis to gluconic acid. The low solubility of oxygen in water at normal conditions would indicate that only a small amount of glucose could be oxidized before oxygen becomes rate limiting. Thus oxygen must be continuously supplied by transfer from a gas phase (air or oxygen) or possibly from H 2 0 2added into the glucose solution. Depletion of oxygen can be important especially in reactor beds and within the catalyst support when its inner part contributes substantially to the reaction. The stability of immobilized glucose oxidase and catalase has been extensively studied, but the experimental conditions usually vary from case to case, making a rational comparison of the used immobilization techniques and carriers difficult. The storage activity has been mainly reported as satisfactory, ranging from one month up to one year with the residual activity higher than 50%; Broun et al.' immobilized GI0 and Cat on cellophane by crosslinking with glutaraldehyde and stored them for one month without the loss of activity. Kunz and Stastny2investigated GI0 bound covalently with y-APTES-glutaraldehyde on zirconia and found the residual activity to be 80% after one year. The residual activity after one year for GI0 bound via isocyanate on nickel oxide higher than 50% was reported by Weetall and H e r ~ h ; ~ and the highest storage stability for Cat adsorbed on titania was given by Kunz and Stastny: 80% of the residual activity after more than one year. The storage temperature usually reported is 4-S"C dry or in buffered solutions. N o matter how important the shelf stability may be, it is the operational stability that decides the economics of any process based on the use of glucose oxidase to produce gluconic acid. Unfortunately this stability is quite low; for different catalyst preparations half-times between 10 and 100 hr are Better
IMMOBILIZED DIFFERENTIAL-BED REACTOR
91
results are usually patented. For example, Forgione7 described G10 and Cat immobilized to diamine crosslinked sulfite carbonyl polymers with only 20% overall activity loss in one month operation. Very good operational stability-up to 60 days-was mentioned by Dinelli and Morisis for their fiber-entrapped G10. Unfortunately very few authors present quantitative expressions for the rate of deactivation. Lesterg studied the kinetics of G10 entrapped in gel and expressed the activity decay in an exponential form. Altomare et al.lo*llshowed that the deactivation of Cat can be described by first-order kinetics with respect to hydrogen peroxide concentration. Furthermore, they found better stability for fungal Cat than that for beef liver Cat. The exponential decay of G10 activity was found also by Gargano6 with the decay constant being a function of the glucose concentration. It is generally understood that the Cat in this double-enzyme system, GIO-Cat, prevents the deactivation of G I 0 by the hydrogen peroxide produced by the reaction, and Bouin et al." showed that there is an optimum for the ratio of these two enzymes. From this and our own experimental evidence we have derived the assumption in our model that the inhibition of G10 might be reversible as long as the active Cat is present in abundance. The present work is a report on the methods used, giving preliminary experimental results and presenting a kinetic model to describe the reaction-deactivation phenomena.
MODEL For the reaction system under consideration, the flow diagram shown in Figure I was suggested. From this the mathematical model with the following assumptions was derived: 1 ) Substrate solution was equilibrated with oxygen or air in a separate step (as in experiment). 2 ) Glucose was always in excess compared to oxygen and its external as well as internal diffusion resistances were neglected. 3 ) Catalase is irreversibly denaturated by hydrogen peroxide in a first-order reaction with respect to peroxide. 4) Glucose oxidase is deactivated by adsorption of peroxide on the active sites according to Langmuir-Hinshelwood kinetics. 5 ) The system is isothermal. 6) Gluconolactone hydrolysis is a first-order reaction. 7) Neutralization of the gluconic acid is assumed to be a very fast reaction. 8) Internal diffusion resistances are neglected. External oxygen transfer is included. Assumption 8 was adopted so that the chemical kinetic formula-
PRENOSIL
92
tion could be qualitatively tested for correct behavior without unnecessarily complicating the formulation. The validity of the model is then limited to very small catalyst particles or the catalyst with enzyme immobilized on.the surface of the support. In a future work this assumption should be relaxed and the effects of internal pore diffusion included. The kinetic model of a batch reactor is the following:
-_
-
k,a[OZB- O,? - R G10,
-dG _
-
- R GlO,
-dL _
-
RG10,
-dGlA _
-
k, L
-dGlo _
- - k,
dt
dt
dt
dt
-
H,O, G10, -
H,Op Cat
k,L
dt
-_ mzoz- R GlO,
+ bk,
+ k,(GIOT - (310,)
k, H,O, Cat
dt
-dCat _ dt
Cat H,O,
- - k,
With the initial conditions at t
0,’
=
=
0:
GIOT = G10,
OzB
G = Go
HZO, = O
L=O
Cat
GIA
=
=
=
G1O0 (3)
Cat,,
0
R is a specific rate of reaction related to the mass unit of the active enzyme given by Lester:9 1 -
R
1
k,G
+-+-1
k,OZS
1
(4)
Rmax
where R,,, is the reaction rate when the glucose and oxygen con-
IMMOBILIZED DIFFERENTIAL-BED REACTOR
93
centration become infinite. A simplification can be achieved assuming the steady-state approximation for hydrogen peroxide:
It gives the following result:
-do's
k,u[OzB - O,? 4R GIOA
-
dt
dG- -
-RGIOA
dt
dL
-=
dt
R GlOA - k, L
dGlA - k, L dt
--
dCat dt
k6 RGlOA k3
The introduction of dimensionless variables:
0," = O,/O,B G10*
=
GlO,/G10,
Cat*
=
Cat/Cat,
P
=
RIRmax
G*
=
G/Go
L*
=
L/Go
GlA*
=
GIA/Go
r =
(7)
tR,,,
The dimensionless rate of reaction is obtained from eq. (4) after making all variables dimensionless:
PRENOSIL
94 P-Glucose
02-Bubbles. Air
1
Absorption lsaturationl dissolved 02 Mass transfer to the surface
dissolved 02
I
H202
Deactivation
4 H202
Hydrolysis
r Deactivation
r Gluconic acid
Fig. 1. Model flow diagram for the oxidation of glucose by immobilized glucose oxidase-catalase.
then do,* - k a -- '(1 d7
dG*
-
O,*)
-
Rmax
-
pG10*
dL* - G10, pGIO* Go
dr
-pGlO* 2028
GO
dr
G10,
-
-L* k7 Rmax
dGlA* - k , L* d~ Rmax
dGIO* d~
_ _ ---
-dCat* _-dr
k, Rmax
-
(1
-
k,G10, (G10*)2 G10*) - -pk3Cato Cat*
kGG1OT pG10* k3Cato
IMMOBILIZED DIFFERENTIAL-BED REACTOR
with initial conditions at
T =
95
0:
o,* = 1
G*
=
=o
G10*
=
1
L*
Cat*
=
1
GlA*
=
1
0
The numerical solution provides the concentrations as a function of time when all the parameters, as in Table 11, are known. EXPERIMENTAL
The kinetics of formation of gluconic acid from glucose and oxygen by use of immobilized enzymes (glucose oxidase4atalase immobilized on pumice particles ( d = 0.4-1.4 mm) and on TiO, beads ( d = 0.2 mm)) were studied in a batch differential loop reactor. The glucose solution (10% glucose by weight) was continuously saturated with oxygen in a sparged thermostated vessel (size -500 ml), pumped through a small differential thermostated reactor containing the immobilized enzyme, and pumped back to the oxygen saturation vessel. The catalyst was packed in this reactor formed by an interchangeable circular capsule ( d = 30 mm, height = 5-10 mm), closed on its ends by Teflon sieves and fixed in a special jacketed vessel. Inlet and outlet of the reactor were perpendicular to the sieves. The entering gas (oxygen or air) was saturated with water vapor in a series of washing flasks. The rate of reaction was measured by automatic titration (Dosimat, Metrohm AG) of the gluconic acid produced. pH was controlled (50.1 pH) in the oxygen dispersion vessel, and the consumption of 1N NaOH used for the titration was automatically registered and processed by the Tektronix Data Aquisition System. All experiments were performed at 20°C. An experiment was interrupted when the overall glucose conversion exceeded 50-60%, and the reactor content was replaced by the fresh glucose solution. No significant change in the reaction rate when the reactor was restarted was observed, except for a short initial period. Depletion of oxygen in the differential bed never exceeded 15% of its saturation value. A similar apparatus for operation under pressure up to 2 atm (202.6 kPa) was constructed. The oxygen used for the saturation and mixing was recycled and only the make-up for its losses caused by the reaction was supplied by a pressure cylinder. The rate of reaction was measured by continuously weighing the titration me-
96
PRENOSIL
Precision pressure gauge
Fig. 2.
Pressure reactor.
dium, which was pumped into the sparged vessel to maintain constant pH. For the recording and processing data, the desk computer Tektronix was again used. Figure 2 shows the apparatus schematically.
ENZYME AND ITS IMMOBILIZATION In all our experiments glucose oxidase (from Merck), 12 U/mg was used. It contained the catalase as a natural impurity; its content was estimated, according to a private communication of the manufacturer, to be 5 wt %. The particles of pumice or TiOz were
IMMOBILIZED DIFFERENTIAL-BED REACTOR
97
coated with a copolymer of phenylenediamine and glutaraldehyde by performing the polymerization in the presence of these particles. The enzyme was immobilized by adsorption: The prepared support was soaked with the solution of enzyme in O.1M phosphate buffer overnight and subsequently washed with the phosphate buffer alone. The catalyst was stored in the buffer solution (pH = 7) at 4°C. The microscopical structure of the catalyst is shown in Figure 3. The dry catalyst particles were imbedded in epoxy resin and ground to show some of them in cross section. It is interesting to see that the pumice carrier had pores large enough to be filled with phenylenediamine-glutaraldehyde copolymer during its polymerization (dark filaments in Fig. 3(a)). On the contrary, owing to its fine pore structure, no copolymer was found inside the TiO, particles. It stayed in the form of a thin coating on the surface (dark areas around the white circles in Fig. 3(b)) indicating, in this case, that the influence of internal diffusion was negligible. RESULTS
The initial reaction rates for the enzyme system bound to pumice and TiO, were measured to preliminarily characterize the catalyst. These experiments were similar to a conventional assay at constant temperature and pH with the only difference that the catalyst was in the differential-bed circulation loop instead of in a stirred tank. Effect of Flow Rate As was expected it was observed that the rate of reaction was a function of flow rate through the reactor. The overall rate of reaction for the system as a whole increased with the flow rate, but the specific rate of reaction measured in terms of conversion per pass through the catalyst bed and unit of time decreased. The conversion in a differential reactor is given by the following equation:
dX - r 1 V Go F The specific conversion versus 1/Fshould yield a straight line with a slope r / G o . The experimental data are plotted in Figure 4. The deviations mainly occur owing to the external mass-transfer resistance, which increases rapidly with a decreasing flow rate. From the initial slope and the known volume of the catalyst V (7 ml/liter), the
98
PRENOSIL
(b)
Fig. 3. Microphotographs of the catalyst imbedded in epoxy resin and ground to show the particle cross sections. Scale = 30 mm is 0.1 mm. (a) Carrier is pumice; dark copolymer well visible inside of the pores. (b) Carrier is TiOZbeads; copolymer on the surface only.
rate of reaction in a hypothetical 1 liter batch stirred-tank reactor (BSTR) was calculated: r' = 4.8 pmol/liter sec. This value, compared with the rate of reaction for the soluble enzyme R = 640
99
IMMOBILIZED DIFFERENTIAL-BED REACTOR
pmol/sec pmol G10 [eq. (4)], gave the estimation of the active GI0 in our catalyst: r ' / R = 7.5 x lop3 pmol/liter. For the simulation calculation its rounded value was used as G10, = lop8 moliliter.
Effect of Gas Composition The initial rates of reaction for air and pure oxygen in the gas phase were measured. The differential-bed reactor (DBR) and BSTR were used in these experiments for comparison with the same charge of the catalyst (P3), so that its absolute amount was irrelevant. The ratio of the initial rates for oxygen and air in the BSTR was found to be R O 2 / R a i=, 1.92. This value is in a very good agreement with the calculations according to the theoretical kinetic expression [eq. (4)] without mass-transfer limitations. Furthermore, because the effect of external mass transfer is supposed to be negligible in the BSTR, we can also say that the influence of internal diffusion for this catalyst must be very small. The absolute values of the reaction rates in the DBR were lower than in the BSTR, but their ratio R 0 2 / R a i was , much higher than in the BSTR and increased for smaller flow rates. It can be explained
I
0
0 01
0.02
003
0 OL
0.05
I / F [min ml-11
Fig. 4. Specific conversion as a function of flow rate. Catalyst is P3; depth of bed = 5 mm; T = 20°C; pH = 6.5; glucose = 0.5M.
100
PRENOSIL
by the presence of significant external mass-transfer resistance in the DBR. The reaction rate for the low flow rates becomes a linear function of dissolved oxygen concentration, being governed by eq. (12) rather than eq. (4),in which case ROB/Rairwould be 4.7. The initial rate ratios can be derived from the slopes of the lines in Figure 5. Catalyst Aging
The most important factor for a successful design of an immobilized enzyme reaction process is the stability of the catalyst. Therefore, a series of experiments to study the performance of the catalyst as a function of time were carried out. The results were plotted as p = R / R o vs. time. R o is the initial rate of gluconic acid formation arbitrarily calculated as the conversion in the first 20 min. As a comparison the stability of the soluble enzyme was studied. A small
Effect of gas composition. Catalyst is P3; T = 20°C; pH 0.5M.(0) Catalyst in BSTR; (0) catalyst in DBR, F = 30 mllmin.
Fig. 5 . =
=
6.5; glucose
IMMOBILIZED DIFFERENTIAL-BED REACTOR
101
amount of G10 was dissolved in the vessel with 500 ml 10%glucose (O.5M). Oxygen was continuously introduced and dispersed by the sintered glass plate. The flow rate of oxygen was high enough to maintain a vigorous mixing. After an initial increase owing to the relatively slow hydrolysis of gluconolactone, the reaction rate decreases as the catalyst is deactivated. In these experiments the effects of dilution and the glucose consumption, which could decrease the rate, were kept small by the relatively high concentrations of NaOH and glucose. Figure 6 shows the typical curve for the soluble enzyme. It is interesting to note that a higher initial enzyme concentration results in a bigger pmax,but the deactivation is faster as well, and after some time both curves are nearly the same. Figure 7 shows these curves for the three different catalyst particle sizes. The initial rise of p is caused mainly by the slow hydrolysis of gluconolactone in this range of pH. The maximum pmaxshifts with respect to the time scale so that for the bigger catalyst particles it occurs later. In the case of soluble enzyme pmaxis reached soonest. This phenomenon suggests the influence of internal pore diffusion on the catalyst particle reaction and activity decay. This influence is probably very small for the catalyst P3 with the smallest particles, as its position of pmaxis almost identical with the flat maximun for the enzyme bound on the surface of titania particles (Fig. 8), and it will be
I
0
5
10
20
15
25 t [hrl
Fig. 6. Deactivation of soluble (310. Glucose
=
0.5M, 0,'
= 1.3mM.
I02
PRENOSIL
rlr,
=
Fig. 7. Deactivation of G10 immobilized on pumice. T = 20°C; pH = 5.5; glucose 0.5M; 0,’= 1.3mM.
shown later that this catalyst should have negligible internal masstransfer resistance.
Effect of Enrichment of Catalyst by Catalase When reaction rate dropped already to a quarter of its pmax,pure soluble Cat (-10 mg) was added into the system. It resulted in an immediate increase in the reaction rate back to its maximum and even higher. This effect cannot be explained by an oxygen “burst” owing to the sudden H,O, decomposition, because this would cause an oxygen supersaturation quickly vanishing in the aeration vessel. In order to study the influence of Cat further, an immobilized G10 enzyme enriched on Cat was used. As the catalyst bound on pumice was showing rather poor stability, another carrier (Ti02) was tried, because it is supposed to be a catalyst for H,O, decomposition. Figure 8 shows its performance as a function of time. It showed a significant increase in the stability as well as in the activity expressed per gram of catalyst (see Table I). The experiment was stopped after 20 hr because of glucose depletion. The experiment when restarted with fresh glucose solution, already showed some deactivation. Addition of Cat had no effect on restoring of the
I03
IMMOBILIZED DIFFERENTIAL-BED REACTOR
1 0-
05 1
=
Fig 8. Deactivation of G I 0 immobilized on TiOZ. (Catalyst is Ti.) T = 20°C; pH 5.5; depth of bed = 3 mm; glucose = 0.5M.
activity. The effect of the Cat addition could be a subject of further study. Effect of Mass Transfer
The reaction rate of the immobilized enzyme in the absence of external mass-transfer and internal diffusion resistance is governed by the kinetic expression for soluble GI0 [eq. (4)]. The precise value of the concentration of the active enzyme is usually unknown and the use of eq. (4) for the calculation of absolute reaction rates is doubtful. However, it can serve for the estimation of relative reaction rates and the mass-transfer influence.
TABLE 1 Catalyst Characteristics
Sample
Initial rate R , Total amount of (related to Particle enzyme for conversion after Maximum rate 20 min) R W X diameter immobilization d (mg enzyme/ml (pm0Vmin.g (pmoVmin.g Support (mm) sediment) catalyst) catalyst)
PI P2 P3 T
pumice pumice pumice TiO,
0.8 0.6 0.4 0.2
10 20 20 30 GIO; I Cat
5.14 37.70 47.70 86.80
8.48 67.10 58.20 98.10
PRENOSIL
104
As is seen in Figure 3 , the titanium particles are coated with only a thin layer containing an active enzyme and therefore intrapellet diffusion is assumed to be negligible for this catalyst. The calculation of the Thiele modulus for spherical particles (@) gives its value to be about 0.2, which fulfills the Weisz criteriont3 for disregarding pore diffusion: when
0.9. To discuss the mass-transfer resistance for oxygen as a limiting substrate, it is necessary to consider only its transport from the bulk liquid to the catalyst surface, because the liquid is saturated by oxygen in a separate vessel prior to its contact with the catalyst. The expression for the external mass transfer per unit volume of reactor can be written in terms of the diffusion rate from the bulk liquid to the surface: R
=
k,u(Oz” - 02’)
(11)
The concentration 0,” may be eliminated by equating R to the rate of the reaction on the catalyst surface as given by eq. (4) and substituting back into eq. (1 1). The rate of reaction results in a complicated quadratic form. From it can be shown that at least in our case the surface concentration O,s is negligibly small and the rate expression becomes simply R
=
k,a 0,”
(12)
In a plot of R vs. O,”, the data should fall into a straight line of slope k , a . The mass-transfer coefficient k , can be estimated using the Chilton-Colburn analogy: j,
=
( k , / U ) ( , U / P D ) ”=~ 1.625 Re,-0.507
(13)
for Re, < 120. The k , was calculated substituting the values of parameters from our experimental conditions into the above equation. It was found to be 0.0057 cmisec for a flow rate in the pressure reactor of 240 mlimin, corresponding to a superficial velocity of 0.57 cmisec. The specific surface area for the spherical catalyst particles d = 0.05 cm and voidage E = 0.26 was estimated to be a = 80 crn2/crn”and therefore k , a becomes 0.45 sec-’. The experimental data were obtained in the pressure reactor as described above. Figure 9 shows the maximum initial reaction rate per liter of reactor volume as a function of oxygen concentration. It was possible to draw a straight line through the experimental points with the slope giving the value for k,a = 0.42 sec-’, which is in very good agreement with the above theoretical calculation.
IMMOBILIZED DIFFERENTIAL-BED REACTOR r [ m m l s-l
105
I~-’I
2-
1 -
2
1
0
3
L
Fig. 9. Maximum reaction rate vs. oxygen concentration. Catalyst is Ti; depth of bed = 8 mm; T = 20°C; pH = 5.5; Re, = 6; k,a = 0.42 sec-*.
Additional experimental evidence for the presence of external mass-transfer resistance comes from the observation that the overall rate of reaction was increasing with the flow rate through the rerlro
I
01
I
0
I
10
20
t ihrl
Fig. 10. Relative activity vs. reaction time. Catalyst is Ti; depth of bed = 8 mm; = 20°C; pH = 5.5; Re, = 8. (0) O z B= 0.27mM, t,,* = 50 hr; ( 0 ) O z B= 1.30rnM, t l l Z= 27 hr; (0) O Z B= 3.90mM, t l I 2= 7 hr. T
106
PRENOSIL
actor, while the specific rate of reaction in terms of conversion per pass through the catalyst bed and unit of time decreased. Effect of High Oxygen Concentrations
As expected, the operation under pressure had an adverse effect on the enzyme stability. Figure 10 depicts the activity as a function of the reactor operation time with oxygen concentration as a parameter. It can be observed that the deactivation is greatly enhanced at higher dissolved oxygen concentration, i.e., higher pressure in the gas phase. DISCUSSION The rate curve for the case of P3 enzyme agrees closely with our mathematical simulation. Especially significant is the coincidence CIA*
G10* Cat*
r [,umol/minl
0.3
I00
0.2
30
50
0.1
:C
20
3
0
Fig. 1 1 .
Results of simulation and experiment. Catalyst is P3.
IMMOBILIZED DIFFERENTIAL-BED REACTOR
I07
of the position of the maximum rate (pmax)on the experimental curve with the theoretical one. The maximum is sensitive to the hydrolytic constant for gluconolactone, which was taken from the literature. l4 The results of the simulation, as compared with experiment, are shown in Figure 11. Although a number of parameters with their numerical values had to be used in the model, only two of them were completely unknown and adjusted by the best fit for the experimental data. Table I1 presents the parameters with the source of their numerical values. Our mathematical model does not include the effect of internal diffusion, but it was felt that its influence compared with that of the external mass-transfer resistance was rather small. The good agreement of the experimental data with the simulation seems to support this assumption. The microscopical investigation predicts smaller diffusional effects in the case of TiO, catalyst. It agrees well with the fact that its maximum rate shifts closely toward the maximum for the soluble enzyme. Oxygen concentration was introduced as a new variable in the pressure reactor. It could be shown that the rate of reaction is of pseudo-first order and so that it increases faster with oxygen concentration than according to the kinetic expression for the soluble enzyme [eq. (4)]. On the other hand, the enzyme is much faster deactivated owing to this higher reaction rate, which makes its perTABLE I1 Numerical Values of Parameters for P3 Enzymea Parameter
Value
0.1 952 1.2 x 10-3 0.5 6x 5 x 10-9 11.944 X lo3 2.15 x lo6 4 x 105 1.5 x 5 x 10-6 1 x 10-3
3 x 10-4 a
Dimension
Source or Ref.
sec-l sec-' moVliter mollliter mollliter mollliter literimol sec literimol sec literimol sec literimol sec sec-' literimol sec sec-*
15 16 saturation value preparation estimated estimated
Glucose solution is 0.5M;pure oxygen.
16 16
17 adjusted adjusted 17 14
PRENOSIL
I08
formance on the whole worse than at the normal pressure or with air. However, the pressure reactor could be used for a fast testing of the stability of different G10-Cat catalyst preparations. Furthermore, the differential-bed reactor can be used for the estimation of the apparent active enzyme concentration in a catalyst that must be known for any design calculation based on the microkinetics of a given enzymatic reaction. Nomenclature U
BSTR Cat d DBR F G GIA GI0
specific surface area of the catalyst (cmz/cms) batch stirred-tank reactor concentration of catalase (mol/liter); abbreviation for catalase in text diameter (cm) differential-bed reactor flow rate (cmYmin) concentration of glucose (mol/liter) concentration of gluconic acid (mol/liter) concentration of glucose oxidase (mol/liter); abbreviation for glucose oxidase in text concentration of hydrogen peroxide (moliliter) kinetic constants mass-transfer coefficient to external surface (cmisec) concentration of lactone (moliliter) concentration of dissolved oxygen (mol/liter) rate of reaction (PmoVliter sec) specific rate of reaction (sec-l), rate of reaction (units in text) particle Reynolds number time (sec, hr) temperature ("C) superficial velocity (cmisec) bed volume (cm3) conversion void fraction effectiveness factor viscosity (gicm sec) density (g/cm3), dimensionless reaction rate Thiele modulus
Subscripts A max 0 T
active maximum initial total
Superscripts
B S
bulk liquid catalyst surface
IMMOBILIZED DIFFERENTIAL-BED REACTOR
109
The author is grateful to Dr. V. Krasnobajew from the Swiss firm Givaudan S A for his assistance in preparing the catalyst and for his guidance during the initial stages of this work. Furthermore, thanks must be given to Mr. R. Carter who carefully carried out some of the experiments.
References I . G . Broun, E. Selegny, S. Vrameas. and D. Thomas, Biochirn. Biophys. Actu, 185, 260 (1969). 2. H. J . Kunz and M. Stastny. Clit7. Cheni.. 20, 1018 (1974). 3. H . H. Weetall and L. S. Hersh, Biochitn. Biophvu. A c t a , 206(1), 54 (1970). 4. K. B. Ramachandran and D. D. Perlmutter, Biotechnol. Bioeng., 18, 669 (1976). 5. K. Buchholz and M. Reuss, Chitniu. 31, 27 (1977). 6. R. Gargano, "Immobilization of glucose oxidase by ion exchange," PhD. thesis, University of Connecticut, 1975 (Univ. Microfilms Ann Arbor, Mich., 48106). 7. P. S. Forgione, U.S. Patent 3 753 681 (1973). 8. D. Dinelli and F. Morisi, Er7zyme Engineeritig (Plenum, New York, 1974). Vol. 11, p. 293. 9. D. E. Lester. "A study of the kinetic behaviour of gel-immobilized glucose oxidase." PhD. thesis, University of Wales (Univ. Coll. Swansea), 1973. 10. R . E. Altomare. J . Kohler, P. F. Greenfield, and J . R. Kittrell. Biotcchnol. Bioetig.. 16, 1659 (1974). I I . R. E. Altomare. P. F. Greenfield, and J . R. Kittrell, Biotechnol. Bioc,t7g.. 16, 1675 ( 1974). 12. J . C. Bouin, M. T. Atallah, and H. 0 . Hultin, Biochini. Biophys. Actu. 438(1), 23 ( 1976). 13. P. B. Weisz. Z . Phys. C h e t ~ .11, , I (1957). 14. R. E. Mitchell and F. R. Duke. At7ul. N . Y . Acud. S c i . . 172, 129 (1970). 15. S . K. Friedlander, AICI7E.I.. 10, 347 (1961). 16. B. Atkinson and D. E. Lester. Biotec.ht7ol. B i o r t z g . . 16, 1299 (1974). 17. R. E . Altomare. thesis (PB 245 096). University of Massachusetts, 1974.
Accepted for Publication April 17, 1978
