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The retention and immobilization of enzymes and cells usually requires the presence of an additional solid carrier phase or flocculant cell mass. As illustrated in Fig 6.1, in order to reach a reaction site, substrate S must first be transported by convection from the bulk liquid to the exterior stagnant film (point A). Then transport by diffusion must occur through the film (from A to B) to the surface of the carrier (point B), where surface reaction can take place. If further reaction sites are available within the carrier matrix, an additional internal diffusion path (from B to C) is then also required. Similarly product P, formed within the carrier matrix, must diffuse out of the matrix towards the surface, and then away from the surface via the external mass transfer laminar film to the bulk liquid.

Diffusion film Concentration

i

B

Bulk liquid

Solid carrier

The stagnant film and the immobilization matrix constitute mass transfer resistances which may slow the overall reaction rate, since reaction cannot proceed at a rate greater than the rate at which substrate is supplied by the mechanism of diffusion. The diffusional mass transfer process via the external film is referred to as external mass transfer. Since the reaction site may often be located within a gel, a porous solid, biofilm or biofloc, the transfer of substrate or substrates from the exterior surface of the biocatalyst to reaction sites, located within the internal structure of the carrier, is also usually necessary. This process is therefore referred to as internal mass transfer or intraparticle transfer. In what follows, external transport and internal transport

Biological Reaction Engineering, Second Edition. I. J. Dunn, E. Heinzle, J. Ingham, J. E. Pfenosil Copyright 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 3-527-30759-1

146

are considered separately, although, of course, the two effects can exert a combined effect in reducing the effective reaction rate, compared to that which would be obtained if there were no diffusional limitations.

6.1

Fig. 6.2 illustrates the substrate concentration profile, existing in the very near region of an immobilized biocatalyst surface, supported on a non-porous carrier. Also shown is the idealized concentration profile, as represented by the film theory. As previously discussed in Sec. 5.2, the "film theory" assumes the presence of a stagnant layer of liquid to exist at the solid-liquid interface. This stagnant region is termed the diffusion film or Nernst-diffusion film and constitutes the external resistance to mass transfer. It thus determines the rate of supply of substrate to the surface, for subsequent reaction.

Substrate cone.

SA A

Figure 6.2. External diffusion model of substrate transport to a reactive enzyme immobilized on a solid surface.

The rate of supply of substrate to the surface is defined by mass transfer considerations, such that the mass flux to the catalyst surface is given by, JS = ks L (SA-S B )

147

where, js is the mass flux (mol/m2 s), ksL is the mass transfer coefficient (m/s) and SA, SB are the substrate concentrations for the bulk and surface conditions [mol/m3], respectively. The steady-state balance can be written for the transport-reaction process, (Rate of supply by diffusion)

k

In the following treatment, the surface reaction is assumed to be first-order, such as found for a biocatalytic reaction with Michaelis-Menten kinetics and S KM- The apparent reaction rate per unit surface area, r app (mol/m2 s), is equal to the rate of both processes. Solving the equation, for the surface concentration, SB,

SB =

and hence

-SA

= ksSB=U^

l k S+ k SL

Two extreme conditions can be identified: 1) For ks/ksL 1, SB approaches zero, and the reaction is completely mass transfer controlled, with rapp = 2) For ks/ksL .! SB approaches SA, and the reaction is kinetically controlled, with an apparent rate equal to that defined by the reaction kinetics, with rapp = ks SAThe intermediate situation is given by the full equation, for which the apparent reaction rate is influenced by both the true kinetic rate constant ks and by the diffusional mass transfer coefficient ksLFor a zero-order reaction: k S L(S A -S B ) = ks where ks is now a zero-order kinetic rate constant. The concentration at the reaction surface SB is thus,

SB = S A -ks/k S L

148

which indicates that the ratio of the magnitudes of the kinetic rate constant to that of the mass transfer coefficient determines SB- If the reaction is zeroorder, the overall order of reaction rate is not influenced by diffusional considerations, but the effective rate will still be reduced, owing to the lowered concentration SBFor Michaelis-Menten kinetics, which encompass the range between effective zero and first order reaction kinetics, the relation between rate of supply and the rate of reaction becomes, kSL (SA - SB) = : > - = r app After rearrangement, the resulting quadratic equation can be solved for SB, with the solution indicating that, in general, the magnitudes of all the coefficients can influence the overall reaction rate and also that the external transfer can change the overall observed reaction kinetics. Thus they no longer follow the Michaelis-Menten form with respect to bulk concentration, and the apparent kinetics can differ substantially from the intrinsic true reaction kinetics. Under these conditions, it is no longer correct to equate the Michaelis-Menten constant, KM, to the substrate concentration at which the observed reaction rate is equal to the half of the maximum observed rate. This can be most easily seen from the above equation; when SB is low, the effective surface rate reduces to the form, rapp = vm SB/KM- The overall reaction rate then becomes,

= app

(v m /K M )S A (v m /K M k S L ) + l

showing that the apparent rate of reaction depends on the magnitude of the mass transfer coefficient It is only possible to measure true reaction kinetics, by operating experiments in a truly kinetic regime, such that any influence of the external diffusional mass transfer is negligible. This can be achieved by ensuring that the ratio of vm/ksL is sufficiently low. Under these conditions,

which are the intrinsic Michaelis-Menten kinetics. The ratio can be made low by increasing the mass transfer coefficient, k$L, and by increasing the mass transfer rate enhancing parameters, such as flow velocity and stirring speed. Conversely those factors affecting the maximum reaction rate, vm, should be decreased, for example enzyme loading and temperature. The regimes of possible external mass transfer influence on the observed kinetics are summarized in Table 6.1, together with the important parameters.

149

Table 6.1. Characteristics of overall reaction rate influenced by external transfer. Regime of operation Transfer control Reaction parameter having an influence on overall rate Temperature (slight influence due to viscosity and diffusion rate). Stirring speed in tank. Flow in packed and fluidized beds. S in bulk liquid. S in bulk liquid. Enzyme loading on surface. Temperature. All of above.

6.2

Reactions with enzymes and whole cells entrapped or immobilized in a porous solid matrix will be subjected to a mass transfer influence. Example systems are whole cells immobilized in alginate, enzymes adsorbed on ion-exchange resins, or naturally occurring biological films on surfaces or flocculated biomass. In the case of a biological film attached to an impermeable solid, the substrate can enter from only one surface, as shown in Fig. 6.1, through the diffusion layer A-B and into the biocatalyst B-C. In the case of an alginate bead, a biofloc or its two-dimensional approximation, substrate can enter from opposing directions, as shown below in Fig 6.3. In this case, the diffusion will result in a symmetrical, steady state concentration profile. The case of complete penetration of substrate through the biofloc is shown by the solid line. Whereas an incomplete penetration, as shown by the dashed line, results in the center of the film being completely ineffective, in terms of reaction capability. Note that in this case, the effects of external diffusion are neglected. The uptake of substrates within solid material requires transport by a diffusional process. The driving force for diffusion is a gradient in concentration, and the diffusional flux is given by Pick's law,

150

jA = -D A

with j having units of kg/m2 s.

dCA dZ

'AO

Diffusion

'AO

Diffusion

'AO

If a reaction occurs within the matrix, a concentration gradient will be established as a result of the simultaneous diffusion and reaction processes. The reaction rate at each position, being usually a function of concentration, will vary, and the overall or apparent reaction rate per unit volume of matrix (kg/s m3), rapp, will be determined by the transfer rate at the surface (kg/s), /Apparent rate\ Vin bulk liquid )

=

Vr a p p = (j|z=o)A = r a v g A L where the units of each term are kg/s. Here ravg represents an average value in the matrix, which will increase with higher internal substrate concentrations. Regarding the influence of diffusion for a particular situation, it is possible to arrive at some quantitative guidelines without considering any mathematical details. Obviously the concentration profiles are caused by a competition between reaction and diffusion. The ratio of the maximum intrinsic reaction rate (not influenced by transfer) to maximum diffusion rate provides a useful dimensionless parameter,

r

151

max A L

r(C 0 ) A L

D (Co/L) A ~

JA

For first order reaction, r = k CQ, this dimensionless group becomes k L2/D, and for zero order reaction, r = k, it is k L2/D CQ. Therefore for any kinetic form of equation, the distance coordinate or length of diffusion path, L, plays an important role since the ratio of maximum diffusion rate to maximum reaction rate varies according to L2. The higher the value of this ratio, the greater in magnitude are the substrate gradients. With this qualitative feeling for diffusion-reaction phenomena, more quantitative aspects can be considered.

6.2.1

Diffusion with biological reaction can be treated by mathematical modelling, and from this it is possible to develop equations describing changes of concentration, with respect to both time and position. The same technique of finite differencing is used as in the modelling of the dynamic behavior of tubular bioreactors, Sec. 4.2.2. Consider the case where the substrate varies from a concentration SQ in the bulk liquid to some concentration, at the position L (a wall or the center of a symmetrical particle). At the center by symmetry or at a wall, owing to the absence of diffusion into the wall, the concentration gradient must be zero. The actual continuous concentration profile, through the slab, may be approximated by a series of increments, as indicated in Fig. 6.4 and by a series of biocatalyst matrix elements as shown in Fig. 6.5.

Liquid

152

jn-1

In

,

I n+1

n-1

>

n+1

A magnified view of element n is shown in Fig. 6.6, where the flux, jn, depends on the local concentration gradient, and the reaction rate, rsn, depends on the local concentration in element n.

Jn-1

AZ

Figure 6.6. A single element n of volume AV and thickness AZ, showing the diffusion fluxes.

A component mass balance is written for each segment and for each component as /Accumulation^ V rate )

=

dSn ~dT = Jn-1 A - jn A + rSn A AZ

A AZ

(Sn-i ~ Sn)

s

n-l ~ Sn)

+rSnAAZ

153

s 5 + rsn

as

a2s

+ rs

The finite-differenced forms of the model equations, however, are especially suitable for simulation programming. Thus, N equations are obtained, one substrate balance equation for each element, and these are solved simultaneously. Note that the boundary conditions, for elements 1 and N, must be described separately. For the above case, the boundary conditions are dS/dZ = 0 at Z = L and S = SQ at Z = 0. Thus the equations for the first and last elements must be written accordingly, as shown in simulation example BIOFILM, Sec. 8.7.1. Note also that it would be also possible, in principle, to include external diffusion effects, by formulating a boundary condition, balance for the first element as: /Accumulation^ V rate /

=

/External trans- A /Diffusion^ f Production \ V port rate in ) ~ \ rate out ) + Vrate by reaction/

where the external transport rate through area A is, Q = k S L (So-Si)A where SR is the bulk reactor concentration.

The biocatalyst diffusion model can be combined with a well-mixed tank model, as shown in Fig. 6.7. The bulk liquid-phase component balances take the form: ^= |(S F -SO)-Jsa| z = 0 where a=A/V,

, A dS , Js a| z=o = - DS v dZ I z=o

where,

154

dS dZ I z=o =

The direction of the positive diffusion is into the biofilm, since dS/dZ is negative and (SR - Si) /AZ is positive. Here SR corresponds to So in Fig. 6.4, and Si is the concentration in the first element. The boundary condition is S = SR at Z = 0.

F,SF

F,SO

Figure 6.7. Coupling the biofilm model to the continuous tank model.

In this way it is possible to simulate immobilized biocatalyst performance in a single tank or in a column by using a tanks-in-series model, The simulation example BIOFILM, Sec. 8.7.1, demonstrates this approach.

6.2.2

There are several advantages of formulating model equations in dimensionless form. The number of variables in the model is reduced, thus reducing the number of experiments or simulations required to investigate all combinations. It is also possible, on the basis of the numerical values of the parameters, to access the relative importance of certain terms. Finally, the dimensionless form makes the solution much more generalized because the units of the individual quantities are no longer important. The governing dimensionless parameters can be obtained by re-examining the defining model equations and arranging them such that the variables range

155

only between the values of zero and unity. Thus new dimensionless variables, S=S/S 0 , Z = Z/L and dimensionless time, f = t / ( L I D ) , can be defined. Substituting these new variables into the diffusion-reaction, partial differential equation, for the case of a first order biochemical reaction, gives,

as

S

(L /D8)at

as

or

at - az2 " DS

_ s

Thus the solution depends only on the value of [lq L2/DsL which is a dimensionless diffusion-reaction parameter. For zero-order reaction the equation becomes,

as a2s2 at " az

where ko L2/Ds SQ is the governing parameter. It is seen that the dimensionless parameters in the model have the same form and significance as was derived from the qualitative reasoning presented earlier. For heterogeneous reaction systems this dimensionless group is known as the Damkohler Number, and its square root is called the Thiele Modulus. In the above equations, all the terms, excepting that of the reaction term, have dimensionless parameters of unity. On this basis, it can be said that if the reaction parameter for a first order reaction, [ki L2/DsL has a value of 1.0 or greater, then the reaction will have a large effect on the solution, that is, on the concentration gradients. Similarly, ko L2/D$ SQ will govern a zero order reaction. Such "order of magnitude analysis" is important for physical understanding and also to obtain information from differential equations without having to actually develop an analytical solution. Dimensionless formulation of equations is also explained in the simulation example VARVOL, Sec. 8.3.1, and KLADYN, Sec. 8.5.5.

6.2.3

The relative influence of diffusion on biochemical reaction rate, can be expressed by means of an effectiveness factor, T|, where,

156

T|

Solutions of the above diffusion reaction model are available in the literature for simple reaction orders (Satterfield and Sherwood, 1963). In Fig. 6.8 the values of t| for zero, first and second reaction order have been plotted against the Thiele Modulus ,<f>, where, O = '\/L 2 kSo n - 1 /D This figure shows that a zero order reaction is not influenced by concentration gradients until the substrate falls to zero in the matrix, corresponding to O > \2 . The other reaction-types are influenced by low concentrations, as the curves for T| indicate. It is seen, for example, that for a first order reaction a value of O = l corresponds to t| = 0.8.

A

A = zero-order B = first-order C = second-order

0 = L(kSo n ' 1 /D) 172 Figure 6.8. Effectiveness factor TJ versus the dimensionless reaction/diffusion parameter (Thiele Modulus O ) for reactions in a flat film with diffusion from one side (after Satterfield and Sherwood, 1963).

157

6.2.4

6.2.4.1

Case A.

For a biofilm or floe, whose oxygen uptake might be taken as a constant (zero order), the corresponding group would be [L2 qo2 Xbi0fiim/D Co2L where qo2 Xbiofiim corresponds to the rate constant k and the oxygen concentration in the outside liquid phase is Co2- Note that Xbiofiim is the biomass per unit of biofilm volume and is not easy to measure. Substituting values obtained from an aerobic biofilm nitrification experiment gives,

2 0

L2 qQ2 X = D C02

For this order-of-magnitude analysis, the value of 1.0 can be used to separate the regions of reaction and diffusion dominance. Thus it is seen if L = 0.1 mm, then the dimensionless group will have a value of 1.0, and it could therefore be expected that a film or floe thickness greater than 0.1 mm would be oxygen limited. From the exact solution, as seen in Fig. 6.8, gradients would appear for a zero order reaction at a value of <&2 = 2.0, instead of 1.0. This example shows how the Thiele Modulus can be used to make useful estimates for diffusion reaction problems, providing rate and diffusion data are available.

6.2.4.2

Case B.

Nitrification reactions, considering only the substrate conversion reactions and ignoring the slow organism growth processes, the reactions can be written as, NH4+ + 3/2O 2 ->

158

The oxygen requirements for the first and second steps can be related to the nitrogen content of NH4+ and NC>2~. These values are si = 3.5 mg 62 / mg NH4+ - N and 82 = 1.1 mg 62 / mg NCV - N. The low yields and low growth rates make it unnecessary to consider growth requirements and kinetics. In previous work (Tanaka and Dunn, 1982) the intrinsic substrate uptake kinetics for the two steps were shown to have a double Monod form for the first step, rNH4 = v m i and for the second step, rN02 = vm2 where v m i and vm2 represent the maximum rates for a particular biomass concentration and the chemical symbols represent concentrations. Considering the diffusion phenomena in the biofilm to be represented by one-dimensional diffusion with quasi-homogeneous reaction, differential balance equations can be written for all reactants and products to describe the concentration profile in the film. Proceeding as described in Sec. 6.2.1, a component mass balance is written for segment n and for each component: (Accumulation^ _ (Diffusion^ _ (Diffusion^ ^ rate ) ~ \ rate *n ) ~ \ rate out J ( Production ^ Vrate ^ reactin J

. K NH4

and the equivalent partial differential equation is obtained by letting AZ approach zero as 3S 32S

Applying this to each component gives the following balances: For NH4+, 3NH4+

= DNH4

a2NH4+

-wo - TNH4

For NO2",

aNQ2~3r

= D

NO2

^2

+ rNH4 - TNO2

159

For NO3%

3N03

For O2,

32N03

3202

" s l rNH4 ~

The stoichiometric oxygen requirements for the first and second reaction steps are given by si and s2. The boundary conditions used represent the bulk liquid phase or reactor concentrations and the zero gradient at the biofilm- solid interface, as discussed earlier. These equations can be written as differential-difference equations using the finite-differencing technique (Sec. 6.2.1). Thus for each of N increments, four component balances will be needed. Three simulation examples, BIOFILM, ENZDYN, CELLDIFF in Sec. 8.7, demonstrate this approach. This system was also analyzed in terms of dimensionless variables. A comparison of the resulting dimensionless NH4+ and O2 balances reveals that, when the second reaction is neglected, the equations are identical if DNH4 = Do2 and if, Q2R where the subscript R refers to the concentrations in the bulk reactor liquid. Under these conditions, to a good approximation, the penetration distances of O2 and NH4+ would be the same. The ratio O2R/NH4+R, which can be varied according to the reactor operating conditions, can thus be used as a criterion to evaluate whether NtLj."1" or O2 might be penetration-limiting. The O2R/NH4+R criterion indicates which component can be limiting, O2 if the ratio is less than 3.5 or NH4+ if the ratio is greater than 3.5. These conditions are not sufficient for limitation, but indicate which component would be limiting. Simulation results from a model that was developed using finite-differencing demonstrates this phenomenon. The profiles in Fig. 6.9, are for the case O2R/NH4+R = 0.07.

160

NH 4 ,NO3,N0 2 (mg/L)

100

0 2 (mg/L)

20

80 60

NO 10

40 20

Figure 6.9. Steady state profiles for constant bulk concentrations showing incomplete oxygen penetration.

Coupling the liquid and biofilm for a batch nitrification reactor as explained in Fig. 6.7, gave the results in Fig. 6.10. Here the influence of oxygen limitation caused the oxygen in the midpoint of the film to rise as the nitrogen substrates were successively consumed.

NH4

100

NO 3, NO2" (mg/L)

0 2 [mg/L]

80 12 64 48 32 16

0

120

80 60 40 20 0

10

8 6 4

2 0

t (min)

Figure 6.10. Simulated biofilm nitrification profiles in a batch reactor. The N-component concentrations are in the bulk liquid. O2 is in the midpoint of the biofilm and indicates limitation during the first 60 minutes.

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