REVIEW

Folia Microbiol. 37 (5), 331-345 (1992)

Physiological Similarity and Bioreactor Scale-up

J. VOTRUBAand M. SOBOTKA

Instituce of Microbiology, Czechoslovak Academy of Sciences, 142 20 Prague 4

Received May 20, 1992

ABSTRACT. An economically effective transfer of biological processes from laboratory to production scale is the main task of
microbial process engineering. In contrast to the principle of geometrical, chemical, thermal, hydrodynamic or chemical simi-
larity, recommended for scale-up of chemical reactors we propose the principle of physiological similarity. According to this
principle same the microenvironment of the living cell must be established to reproduce the same physiological function (e.g.
growth, product formation or substrate consumption rates) in the large scale bioreactor as in the laboratory one.

CONTENTS

1 Introduction 331
2 Physiological homogeneity 333
3 Physiological similarity 335
3.1 Physical concept - dimensional analysis 336
3.2 Biochemical concept - phenomenological modeling 339
References 342

List of symbols

a exponent in rate equations (see Table II) a, fl coefficients of interface heat and mass transfer
b birth rate p density
c specific heat /* viscosity
C concentration a surface tension
d death rate or impeller diameter V Laplace operator
D diffusion coefficient or diameter A difference operator
g gravity constant
k, K kinetic constants (see Table II)
kLa aeration capacity
L length
n rotation speed
p, P pressure (quantity and dimension)
R reaction rate
SR reaction heat
v, V velocity (quantity and dimension)
Y yield coefficient

1 Introduction

Attempts to transfer a certain biotechnological process to a higher production capacity are

governed by the economy of scale. Collections of cost data for both individual equipment and whole
plant construction have given rise to the so-called six-tenth factor (Hacking 1986, Reisman 1988) which
means that, if a plant capacity is doubled, the cost will be only 20.6 higher. The second reason for scall-
ing-up is that the operating costs, based on production plant volume, decrease with the exponent in the
range of - 0.3 to - 0.5 (Anonymous 1979; Ulrich 1984). Generally, the scale-up of multiphase chemical
processes from the laboratory to the industrial level is based on the knowledge of reaction kinetics, its
changes caused by physico-chemical factors the thermodynamic of the process, and by the character of
m L ~ g and interface mass transfer ( H o r ~ et aL 1980). Except for heterogeneous catalytic reactions,
the changing size of the apparatus can be assumed to be reflected merely in hydrodynamic relations,
the local mechanism of chemical kinetics being unaffected by the scale-up (Marculevich et aL 1984).
The invariant microkinetics of chemical reactions then make it possible for a scale-up through geomet-
332 J. V O T R U B A and M. SOBOTKA Vol. 37

ric similarity as well as through similarity of mass, energy or momentum transport (Himmelblau and
Bischoff 1968). The theory of scale-up hinges on the following methods and rules that make it possible
to transfer information obtained in the laboratory to the industrial level (Hartmann et al. 1984).
1. Gradual transfer and refinement of information via a number of devices with different volumes
(scale-up line or scale-down techniques).
2. Computation-graphical interpolation end extrapolation without special regard for physico-chemical
laws (regression models).
3. Application of the theory of geometrical similarity.
4. Simulation by means of mathematical deterministic models that involve the physico-chemical laws of
the process in a simple form.
5. Application of the possibility theory to handle the uncertainty in the data (Dorochov and Kafarov
1989; Dubois and Prade 1987);
6. Solution of phenomenological equations describing in detail the interrelationships between all the
variables associated with the process (Clementi et al. 1988).
The conventional device of fermentation technology is the aerated, mechanically stirred reac-
tor. A standard stainless steel tank fitted with baffles and sterile protection, air sparger and turbine
impeller is a device characterized by considerable technological versatility; in pharmaceutical and food
industries a switch of organism and cultivation medium makes it possible to change from one type of
production to another without any substantial changes in machinery. It is rather remarkable that the
earliest fermentors for antibiotic production (constructed in the late 1940's and early 1950) remain
usable today for many generations of new microbial products (Reisman 1988). According to this
author, "... a stirred tank gas-liquid contractor has endured for four (!) decades; there is no indication that
the design will be soon retired".
The volume of the production device may range from several liters to hundreds of cubic
meters. This depends on the balance between demand for and supply of the product on the market
(Hacking 1986); indeed, the size of the production fermentor for a new antibiotic or for a micro-
biological transformation may be one-tenth or less of the volume of an inoculation tank for the
production of SCP, baker's yeast, citric acid or penicillin. Scale-up is usually accomplished in discrete
stages, involving several laboratory and pilot plant scales of operation. These four stages should corre-
spond to the following four scales of operation:

(I) shaken flasks (50-1 000 mL)

(2) laboratory scale (5-20 L)
(3) pilot plant operation (50-5 000 L)
(4) production scale (10-1 0t30 m 3)

Transfer of microbial technology from the laboratory to the industrial production level is criti-
cally affected, in contrast to chemical reactors, by the physiology of growth and production, i.e. by the
relationship between the potential production ability of selected microorganism and the external con-
dition in the bioreactor. The local microldnetics of growth and product formation depend both on the
hydrodynamics of the device and the previous history of the culture (Votruba and Van6k 1989).
Interference with the physiology of production during the initial phase of cultivation, such as
a short interruption of aeration between the 6th and 12th hour of cultivation of Streptomyces aureo-
faciens~ is reflected in a drastic drop in the final production of tetracycline after a cultivation lasting for
several days (Matelov~i et at. 1955). A number of similar examples are known from the technology of
microbial processes (Finn and Fiechter 1979; Van6k et al. 1981); failure to respect the physiology of
microorganisms is the most frequent cause of failure in the scale-up of seemingly simple technologies,
such as the production of SCP (Vardar 1983) or conventional waste-water treatment (Aivasidis and
Wandrey 1990). While in chemical reactors mechanical stirring in multiphase systems ensures
(a) dispersion of gas and liquid and/or dispersion of immiscible fluids,
(b) suspension of solids throughout the volume of the reactor, and
(c) homogenization of soluble substances and draining of reaction heat,
in biotechnological reactors the mixing also must ensure the biochemical and physiological homogene-
ity of the microbial culture.
1992 PHYSIOLOGICAL SIMILARITYAND BIOREACTOR SCALE-UP-review 333

2 Physiological homogeneity
can be envisaged as a dynamic equilibrium state (yon Bertalanffy 1953) between the biological activity
of the living cell and the environment. Each deviation from this equilibrium caused by environmental
change brings about a stress reaction that results in disintegration of physiological functions. Their
restoration requires energy which the microorganism draws from the substrate. Ultimately, this causes
a lower yield.

ENVIRONMENT

PHYSICAL CHEMICAL BIOLOGICAL

- TEMPERATURE - p H & REDOX POTENTIAL - PHAGE INFECTION

- PRESSURE - BUFFERING CAPACITY - CONTAMINATION
- SHEAR - SUBSTRATE CONCENTRATION - INHIBITORS
- SURFACE TENSION - PRECURSORS
- VISCOSITY - GENETIC STABILITY
- DiFFUSIVITY - INOCULUM
(SIZE & QUALITY)

POPULATION I
PHYSIOLOGICAL STATE

PHYSICAL

- MORPHOLOGY
-
CHANGES

PIGMENTATION ,//
PRODUCT
BIOCHEMICAL

- CELL COMPOSITION
- ENZYME ACTIVITY
CHANGES

Fig. 1. The factorsinfluencingthe physiologicalstate and physiologicalsimilarityin a bioreactor.

Fig. 1 schematically illustrates the relationships between the environment, the microorganism,
and individual factors that can affect the yield. The factors affecting the physiological state (M/dek
1982; t~i~ica and Votruba 1982) of the culture can be divided into physical, chemical and biological.
Among the markers of the physiological state one may cite morphological traits (Schmidt 1983) or
variability of biochemical composition of the producing organism (Fredrickson et al. 1967).
Physical factors, such as pressure, temperature, pH or redox potential of the broth affect the
kinetics of growth and production (Votruba and Vanrk 1989). In contrast to chemical reactions, the
temperature interval in which microorganisms grow and produce the required substances is relatively
narrow. The content and composition of proteins and, hence, the biological activity, changes markedly
with temperature and hydrostatic pressure (Onken et al. 1984).
In a large bioreactor the physiological nonhomogeneities may be expected to arise because of
insufficient heat removal (Nagatani 1971; Hamer and Heitzer 1990) and variation in the hydrostatic
pressure during mixing of fermentor contents. Lejsek and Kahler (1990) observed that these effects
were important for large-scale beer fermentation (250 m 3) in cylindro-conical tanks.
A considerable source of physiological nonhomogeneity is the impeller itself; in its dose vicin-
ity shear forces cause mechanical stress and damage to cells (Ujcovfi et al. 1980; van Suijdam and Metz
1981; Bronnemeier and Markl 1982). This effect is most often observed in the cultivation of filamen-
tous microorganisms in which it bears a direct relationship to production (Ettler 1990; Bushell 1988)
but also in yeasts, most of which have the hydrodynamically ideal shape of an ellipsoid of rotation. We
334 3. V O T R U B A and M. SOBOTKA Vol. 37

found that in this case mechanical mixing can affect cell budding and interfere with the cell cycle
(Vran~ et aL 1982).
Another source of physiological nonhomogeneity is foam formation caused by changes in sur-
face tension brought about by cell lysis or by production of surfactants. During foaming a fraction of
the microorganisms float in the foam (Sobotka et al. 1981) and their concentration there exceeds sev-
eral times that in the fiquid phase. In the foam the substrate is rapidly exhausted, growth ceases and the
cell may lyse. Even when some of the ceils are gradually dispersed from the foam back into the liquid
phase rich in substrate, the alternation of starvation and nutrition intervals brings about changes in cell
composition (Vranfi et al. 1981, 1982, 1985; Minkevich et aL 1990) and in physiological activity and
increase substrate consumption. Such changes affect most often the biosynthesis of secondary metabo-
rites and are reflected in declining production. On the other hand, when the interval of foam hold-up of
microorganisms is effectively shortened (short mixing time), both foam formation and flotation have
a positive effect on the total rate of oxygen consumption (Sobotka et al. 1981).
The changing viscosity of the broth (Charles 1978; Hubbart 1987; Singh et al. 1990) during cul-
tivation can also cause the physiological nonhomogeneity as it leads to changes in the character of
mixing which gives rise to "deadwater zones" (Wittek and Markl 1978; Hamer and Heitzer 1990). Their
effect on growth physiology and product formation is similar as the effect of foam described above.
Chemical substances affecting the physiological state of the culture act mostly through local
concentrations of compounds participating in chemical reactions and also in a complex equilibrium
among biopolymers, metal ions and other chemically ill-defined components of the cultivation broth.
In the scale-up of animal cell cultures with monolayer cultivation systems (Spier and Griffiths
1985) the main problem is to fred a cheaper medium than in the laboratory ("medium scale-up"). Such
a medium must contain the nutrients and the other, apparently inert, components that are able to
buffer either the substrate-induced acidification or to adsorb other inhibitory products of metabolism,
e.g. ammonium ions and peroxides (Iio et al. 1985; Lubbe et aL 1985).
The complex acid-base equilibrium affects the transport of ionized molecules between the
cell and the medium. The buffering capacity of the medium and concentration of hydrogen ions in the
medium (Minkevich 1979), which change during oxidation of organic substances contained in the
medium, affect the intracellular pH profile and thus also the activity of the enzyme systems of the pro-
ducer organism and also the rate of diffusion of ionized molecules in the electrical field of the cell. The
mapping of intracellular pH performed by a fluorescence method (Slav~ 1982) showed that several
minutes of starvation of cells brings about a prolonged change in the electrical field of the cells even in
such relatively refractory microorganisms as yeasts. The pH value influences the maintenance demand
for sugar in penicillin fermentation. With pH kept at 6.5 the high-yielding Panlab strains have an aver-
age maintenance demand nearly 30 % less than other strains (Mou 1983).
An important source of physiological nonhomogeneity can be the material used for the con-
struction of bioreactor (Baker 1973; Cooke and Williams 1967). The ions, mainly inorganic acids
(sulfuric and hydrochloric) and organic acids in the medium can cause the electrochemical corrosion of
stainless bioreactor body. Copper and copper alloys should be avoided in fermentation applications. All
parts of the vessel should be made of the same grade of stainless steel to minimize electrochemical cor-
rosion. Materials of bioreactor construction in breweries are discussed by Irving (1968). It is known
that complex ion equilibria can cause ion exchange among the metal components of the bioreactor
body (mainly of agitator bearings) and thus change the composition of trace metal ions present in the
original medium.
Among the chemical compounds present in the broth the physiological role of gases (oxygen
and carbon dioxide) is very important in the bioreactor scale-up. In a review paper, Crueger (1987)
summarized the observed effects of oxygen and carbon dioxide on microbial physiology. Generally,
oxygen is toxic to all organisms but the sensitivity varies. The critical concentration ranges from almost
zero for strictly anaerobic species to about 0.1 MPa partial pressure, which is the common maximum
value for most aerobic species. Thus the oxygen toxicity, as a source of physiological nonhomogeneity,
should be taken into account when very tall fermentors are used. On the other hand, many specific
reactions in living cells are limited by oxygen. The critical oxygen concentration for most aerobic
microorganism is about 10/zmol/L corresponding to 4 % air saturation. If no oxygen is available
a facultadve organism turns to anaerobic metabolism. A short interruption of aeration, e.g. in A c e t o -
bacter aceti, results in an immediate production drop and changes in the morphology of the producer
cells (Muraoka et aL 1982). A similar effect was observed for secondary metabolite production, e.g.
tetracycfines (Matelov~ et aL 1955), penicillin (Crueger 1985) etc. Carbon dioxide is required by all
microorganisms, because it is utilized in carboxylation reactions. Especially when cells are grown in
1992 PHYSIOLOGICAL SIMILARITY AND BIOREACTOR S C A L E - U P - review 335

simple salts media without complex nitrogen sources it is observed that low carbon dioxide concentra-
tions are required. Like oxygen, carbon dioxide always becomes inhibitory at increased concentrations.
An extensive review of the inhibitory effects of carbon dioxide has been published by Jones and Green-
field (1982).
Among the biological factors affecting physiological homogeneity some defects may be caused
by the sterile barrier separating the cultivation chamber from the ambient environment or phage infec-
tions of the producer strain.
A separate chapter in the problems of scale-up is represented by new genetically constructed
strains; in this case, the scale-up is complicated by limited stability of artificially produced recombinants
(Imanaka 1982; Young 1984; Schwab 1988; Prokop et al. 1991). The stability of recombinant micro-
organisms is very sensitive to environmental conditions, especially temperature and oxygen concentra-
tion (Schwab 1988). Oldshue (1983) showed that the shear stress produced by the agitator may be the
most important factor in a successful scale-up of recombinant fermentations (Naveh 1985; Yao et aL
1990). The large scale manufacture (2 m 3 airlift bioreactor) of recombinant antibodies involves taking
a vial of cells to an appropriate inoculum volume and the skill of scale-up is mainly in establishing sta-
bility of cell lines over the required number of generations (Bright et al. 1991).
At the end of this section an important source of physiological nonhomogeneity must be men-
tioned. It concerns the volume and quality of the inoculum. Aivassidis and Wandrey (1990) showed that
"crucial to a successful operation o f the large-scale system is the ability to generate adequate amounts o f
preadapted biomass that can be used to inoculate and start the large bioreactor system". This scale-up
study concerned a conventional anaerobic wastewater treatment plant of 1 200 m 3 working volume.
The scale-up ratio for inoculum transfer was 1:200. The transfer of inocululn to the production bio-
reactor represents a tremendous physiological shock. It can reproducibly survive only in a part of
microbial population. Therefore, in most technologies where biologically active compounds are pro-
duced, the scale-up ratio for inoculum transfer is not larger than 1:20. Ritterhaus et al. (1989) in
a series of papers concerning large-scale fermentation of plant cells in an airlift bioreaetor showed that
the ratio of 1:10 for inoculum transfer was sufficient for seed and pilot tanks up to 7.5 m 3 and it must
be lowered to 1:5 when the inoculum is transferred to a production vessel of 75 m 3 working volume. In
his review, Kennedy (1984) pointed out that the majority of scale-up problems fall into three main cat-
egories: (a) development of inocnlum, (b) medium and system sterilization, and (c) aeration and agita-
tion. The bioengineers understand the last two, while the first remains somewhat mysterious.

3 Physiological similarity

According to the principle of physiological similarity we assume that the microkinetic behavior
(physiological functions) of living cells will be similar in the lower-scale bioreactor and in the large-
scale one, when we establish the same microenvironmental conditions for the development of living
cells.
As we mentioned in the previous paragraph, the scale-up ratio 1:10 is taken to be the upper
acceptable limit in a gradual empirical transfer of new biotechnological processes up to 100 m 3 working
volumes. By comparison, the scale-up ratio in some reactors in pharmaceutical industry ranges from
1:10 to 1:4200 (Veksler et al. 1984) and a ratio 1:10 is taken to be the lower limit. Obviously, a gradual
scale-up of long-term fermentations in a 1:10 scale-up ratio is time-consuming and hard to accept. To
shorten the necessary interval and, at the same time, to eliminate the risk of scale-up, antibiotic indus-
tries have built expensive computer-linked systems, in which fermentations conducted simultaneously in
fermentors of different size (3, 30, 300 and 3 000 L) serve to assess the effect of bioreactor volume on
the course of biosynthesis (Knorre et al. 1983). Such a line, which largely eliminates random effects
caused by the changing quality of raw materials and inoculum, makes it possible, on the basis of objec-
tively monitored data, to develop regression extrapolation models for a rapid scale-up of production of
desired antibiotics, and is thus also economically feasible, as was shown for nourseothricin by Weide et
al. (1987).
The scale-up criteria based on the principle of physiological similarity can be broken up into
two area (Aiba et al. 1973; Atkinson and Mavituna 1983; Lilly 1983; Rehm and Reed 1986; Moo Young
et aL 1985; Einsele et al. 1985). The first approach uses the physical concept of dimensional analysis, i.e.
keeping similar geometry of bioreactor, similar boundary and/or initial conditions and important
dimensionless criteria (e.g. Himmelblau and Bischoff 1968) constant. The second concept uses mathe-
$36 3. VOTRUBA and M. SOBOTKA Vol. 37

m a t i c a l m o d e l s to simulate the fluid flow a n d b i o c h e m i c a l reaction in the vessel with a p p l i c a t i o n of

c h e m i c a l r e a c t o r t h e o r y ( L a p i d n s a n d A m u n d s o n 1978).

Table L Development of and relation among dimensionless groups in bioreactor scale-up

MASS BALANCE Rate of Rate of Rate of Generation Rate of

(per unit change of change of change of of component interphase
of volume) mass in mass by mass by by reaction transport
time convection diffusion

Product ((71) OC1/Ot +V(Clv) = DV2C1 +Rp +flACl

Substrate ((72) OC2/Ot + V(C2v) = DV2C2 -Rs +flAC2
Biomass (C3) OC3/Ot + V(C3v) = DV2C3 (b - d)C3 -
Dimensions - VC/L DC/L 2 R flC/L

av LV/D Bo Bodenstein -'~

Y
Yield Y Se Schmidt/~/D~

"r RL/VC Da I Damk6hler ~-

)-------- ~L/D Sh Sherwooa 4

MOMENTUM Rate of Rate of Rate of Generation Pressure Rate of

BALANCE change change of change (gravity) interphase
(per unit in time transport by molecular
of volume) by convection transfer

oq,,,)/ot + v(pw) = v~, +pg - Vp - v~

f~
Explanation "inertial forces" viscous external pressure surface
forces forces gradient tension
Dimensions pv2/L ~,V/L2 pg P/L alL 2

LVp/t~ Re Reynolds

-It V2/gL Fr Froude

) p/V2p Eu Euler Pr Prandtl Cpl~/~

~ pV2L/o We Weber t

HEAT BALANCE Rate of Rate of Rate of Generation Rate of

(per unit change change change of heat intephase
of volume) in time by convection by conduction by reaction transport

o~oCvTO/ot +V(pCpTv) = ~V2T +SR +aAT

Dimensions pTVCp/L 2T/L 2 SR aT/L

) aL/~, Nu Nusselt
VcpL/2 Pe Peclet - - (
)-'----- SRL/VcpT Da II Damk6hler '~

3.1 Physical concept - dimensional analysis

I n d e a l i n g with t h e influence of mass, m o m e n t u m and e n e r g y t r a n s p o r t on m i c r o b i a l physiol-

ogy we c a n n o t always evaluate all t h e i m p o r t a n t effects in an explicit form. A m o r e convenient
a p p r o a c h is the construction o f relationships b e t w e e n the m e a s u r e d variables b a s e d on the t h e o r y of
similarity. F o r two systems g o v e r n e d b y t h e s a m e differential b a l a n c e s of mass, h e a t (energy) a n d
m o m e n t u m (see T a b l e I) w e can s u p p o s e a similar solution, w h e n t h e systems have a similar g e o m e t r y
a n d the b o u n d a r y a n d initial conditions expressed in dimension-less form a r e the same. W h e n all the
1992 PHYSIOLOGICAL SIMILARH~AND BIOREACEOR SCALE-UP-review 337

important dimension-less groups are equal one can expect high reliability in extrapolation to large
scale. These relationships among dimension-less groups can be readily used for the estimation of coef-
ficients that characterize the rates of heat and mass transfer, power input and kinetics of mbdng
expressed as mixing time necessary for full homogenization of cultivation broth which is a complex
mu~fiphase system. Following the above def'med principle of physiological similarity, we have to com-
pare the rates of physical processes with apparent physiological functions which are also expressed as
rates. Physiological homogeneity is the necessary condition for that comparison, because it permits to
achieve the desked dynamic equilibrium among mass and energy flows between the riving cell and its
close environment. Only comparable rates of physical and physiological processes are important for
biereactor scale-up. For example, when the rate of oxygen transfer on pilot scale and on production
scale is ten times higher than the oxygen uptake rate by microorganisms, the scale-up according to their
aeration capacities cannot be used because of lack of oxygen limitation. In engineering practice, we
wish to select dimensional groups and geometrical simplexes, essential for characterizing the relations
among rates of exchange of mass, energy and momentum in the bioreactor.
Einsele et aL (1985) evaluated the geometrical simplexes characterizing different industrial
fermentors. He summarized his findings into the three following rules: (1) The ratio of impeller dia-
meter to that of the bioreactor changes from 0.3 to 0.38; (2) the ratio of fermentor height to its diame-
ter is up to 2; (3) the fermentor has 2 or 3 impellers.
All other important dimensional groups can be developed either by Buckingham's at-theorem
or by inspection (Hk'nme|blau and Bischoff 1968). The application of z~-theorem to mechanically agi-
tated reactors makes it possible to reduce the number of process variables to the minimum number of
dimension-less group. The details may be found in basic books on mixing (e.g. St6rbfi~ek and Tausk
1959; Strenk 1975). As shown in Table I, one needs at least 12 basic dimension-less groups to describe
the systems under scale-up to respect the mass, energy and momentum conservation laws. Evidently,
this is too much and therefore one must exclude, by careful "inspection", the groups that are not
important for the given case (Hhnmelblau and Bischoff 1968). For example, when the bioreactor is
perfectly mixed one can exclude Bodenstein, Froude, Weber and Reynolds numbers. For this example
one can correlate biomass and product 3field with Euler and Sherwood numbers. When the process is
supposed to be isothermal one can exclude Nusselt, Peclet and Prandfl numbers from the analysis.
Experimentally determined correlations among dimension-less groups that are summarized in
TabIe I can be extracted from engineering literature for a number of different geometrical
configurations (e.g. Streak 1975). New studies concerned with novel technological variants appear at
a rate of several tens per year. The main problem is to select from this flood of data and possible
geometrical configurations a suitable function among the dimension-less groups that would have
a causal relationship to the rate of product formation. As a guide one can use the cost analysis of the
process (van Brunt 1986; Reisman 1988) or the preservation of physiological homogeneity of the
producer culture which includes both the engineering and the biological aspects of the technology.
Seichter et aL (1990) showed that for the working volume of a classical, mechanically stirred, bloreactor
lower than i m 3 the influences of boundary effects such as surface aeration etc. are so high and
therefore the engineering data on aeration capacity cannot be used for extrapolation to higher volumes.
With cheap products, such as bacterial or yeast single-cell protein, an important role in the
economy of the process is played by the consumed energy (Moo Young et aL 1979); the process can
thus be scaled-up according to the well-known power input, i.e. the Euler number, in correlation with
the Reynolds number and the Froude criterion (e.g. Fukuda et al. 1968; Aiba et aL 1973; Strenk 1975).
For viscous media typical of filamentous microorganisms or the production of polysaccharides,
when the type of mixing shifts during the fermentation from a fully turbulent state to laminar flow, the
possibilities of this approach are restricted and the requirements of physiological homogeneity are
imposed during the scale-up especially in relation to the effect of shear forces near the impeller on the
producer organism or to limitation by dissolved oxygen. Volume homogenization in relation to oxygen
can be attained in these systems mostly by increased aeration, while mechanical mixing ensures an
effective dispersion of the gaseous phase (Biryukov et aL 1973; Hubbard 1987). The last author summa-
rized his experience in two recommendations about the scale-up procedure for a bioreactor containing
non-Newtonian broths.

Method I
a. Determine the volumetric gas flow rate Q, using the condition that the volume of air per volume of
bioreactor per rain or gas superficial velocity is constant.
 J. VOTRUBA and M. SOBOTKA Vol. 37

b. Calculate the rotation speed N from the power correlation and the aeration capacity kLa from cor-
relations suitable for non-Newtonian fluids (e.g. Yagi and Yoshida 1975; Charles 1978; Ogut and Hatch
1988).

Method H
a. Determine the rotation speed N from the condition that the product of rotation speed and impeller
diameter is constant.
b o Calculate the gas flow rate from the power correlation and aeration capacity kLa from correlation
suitable for non-Newtonian fluids.
As shown by Koloini (1989), at a Reynolds number larger than 1 000 (the usual case in indus-
trial conditions) only a moderate change in heat transfer can be expected due to aeration.
The correlation of aeration capacity (Sobotka et aL 1982) with the parameters of the bioreac-
tor may be, as shown in Table I, described by the relation among the Sherwood, Reynolds and Schmidt
numbers (Aiba et al. 1973; Sherwood et al. 1975; Kafarov et al. 1979; Sokolov and Domanskij 1976).
Analysis of our own data for a nonconventional fermentor, with a design preventing foaming,
was conveniently done via a correlation between the criteria derived from the mechanics of hetero-
geneous flow (Nigmatulin 1978), which has the form of a relation between a modified Stanton number
(St = S h / B o = kLa/n) and the aeration Weber ,number (We = Vg D g p / o ) that makes it possible to
estimate a change in aeration capacity as a function of changes in the surface tension of the broth.
Einsele et al. (1976, 1978) compared the data from 30 conventional bioreactors of 0.1-300 m 3
volumes that had been empirically optimized for production of biomass and secondary metabolites.
Increasing volume was found to be paralleled by decreasing specific power input per unit volume P / V
V -0"37. The value of specific power input for large-scale bioreactors up to 10 m 3 of working volume
ranged from 0.8 to 3 kW/m 3. The homogenization time Tmix increased with bioreactor volume with
V -~ For large-scale bioreactors the homogenization time was about i min. The aeration capacity kLa
lay in the range of 200-800/h, which ensures sufficient biomass growth. The most surprising feature of
the analysis is the fact that in most bioreactors the stirrer tip velocity n'd'3r was practically constant and
ranged from i to 3 m/s. Based on the empirical experience of process engineers in microbial industry,
summarized by Einsele et al. (1976), we can recommend the assumption of a constant Stanton number
for the scaling-up procedure.
Evaluation of the data revealed that the Stanton number defined above has to be modified by
a geometrical simplex which characterizes the ratio of bioreactor body and impeller diameter. Finally,
we can formulate the rule that group kLa'Dreactor/(n'd) has to range from 0.05 to 0.2. The modified
Stanton number used here includes both important macroscopical effects on microbial physiology and
physiological homogeneity, macromixing and oxygen transfer. The data published by Ogut and Hatch
(1978) support this view.
When cultivating the cells that grow in the form of filaments the stirrer tip velocity may cause
their damage and thus affect the production (Asal et al. 1978). Kossen and Metz (1976) found experi-
mentally that the length of the hyphal filament was inversely proportional to the stirrer tip velocity
rinsed to 0.5-1.5 and also to the energy dissipated by the impeller and by the aeration powered to 0.25.
These two independent studies (Einsele 1976; Kossen and Metz 1976) confirmed that the requirement
of physiological homogeneity and physiological similarity may be dominant for successful scale-up of
processes exploiting filamentous microorganisms, especially actinomycetes, for production of biologi-
cally active species (Votruba and Van6k 1989).
In cultivations of pellet-forming filamentous microorganisms cells of the producer are more
resistant to mechanical damage (Blanch and Bhavaraju 1979) and the diameter of the pellet is inversely
proportional to the stirrer tip velocity raised to the power of 0.3 (Suijdam and Metz 1981). From
a mechanical viewpoint the cultivation of pellets would thus seem to be more advantageous but in fact
the formation of pellets brings about local physiological nonhomogeneities of the producer organism
caused by the diffusion of oxygen within the pellet (Bhavaraju and Metz 1975). The effective factor
reflecting the internal diffusion in a pellet 10 mm in diameter is 0.99 for a carbon substrate but a mere
0.2 for oxygen (Kobayashi et aL 1975). The characteristic dimension for scale-up is the pellet diameter
or thickness of microbial film (Atkinson 1974).
1992 PHYSIOLOGICAL SIMILARITY AND BIOREACTOR SCALE-UP - review 339

1o0 !

1
Y

10

4,5
e,7

0.1

7"I

0.01 I I \ I ~d,
1 10 100 10o0
x

Fig. 2. Summary of scale-up criteria;

x - ratio of bioreactor volumes V2/V1 or impeller diameter cubed (D2/D1) 3
y- ratio of power input per unit of volume (P/V)2/(P/V)I
1- equal heat transfer per unit of volume, laminar flow
2 7 equal blend time, turbulent flow
3 - equal Froude number, or equal heat transfer coefficient on stationary surfaces, turbulent flow
4 - equal bubble and drop diameter, turbulent flow
5- equal heat transfer coefficient, laminar flow
6- equal blend time, laminar flow
7- equal mass and heat transfer coefficients for particles bubbles and drops, turbulent flow
8- solid suspension, turbulent flow
9- equal tip speed, turbulent flow
10 - equal tip speed, laminar flow
11 - equal Reynolds number, turbulent and laminar flow

Fig. 2 s u m m a r i z e s the scale-up criteria a n d rules a d a p t e d a c c o r d i n g to K e n n e d y (1984). T h e

g e o m e t r i c scale-up rules a r e p r e s e n t e d as the relation of p o w e r input r a t i o o n r a t i o of vessel v o l u m e s or
i m p e l l e r d i a m e t e r s cubed. N o t i c e that the flow r e g i m e (i.e. either l a m i n a r o r t u r b u l e n t ) greatly affects
t h e p o w e r p e r unit v o l u m e r e q u i r e d .

3.2 B i o c h e m i c a l concept - p h e n o m e n o l o g i c a l m o d e l i n g
A s shown in Fig. 2, the application of the t h e o r y of g e o m e t r i c a l similarity in t h e scale-up of
b i o r e a c t o r s has only a tentative c h a r a c t e r and serves as a basis for scale-up d e v e l o p e d on highly simpli-
fied m a c r o s c o p i c m o d e l s such as in chemical r e a c t o r s (e.g. H a r t m a n n et al. 1979). A s s u m m a r i z e d in
Fig. 2 t h e d i m e n s i o n l e s s groups a r e derived f r o m a particular f o r m u l a t i o n of m a s s m o m e n t u m a n d
e n e r g y (heat) balances, w h e r e the relationship a m o n g t h e m is e s t i m a t e d a n d p r o v e d b y e x p e r i m e n t s .
T h e b i o c h e m i c a l concept of scale-up differs f r o m that above, b e c a u s e it uses the simplified
m a t h e m a t i c a l f o r m o f b a l a n c e s with r a t e t e r m s formally expressed as b i o c h e m i c a l o r e n z y m e reactions.
T h e simplified empirical r e g r e s s i o n m o d e l s (e.g. D a n g et al. 1975) of the "black b o x type" a r e p o p u l a r
 J. VOTRUBA and M. SOBOTKA VoL 37

for the design of automated control systems (e.g. Zudin et aL 1987; Cadman 1991) but their extrapola-
tion abifity for scale-up of bioreaetors is very low (Biryukov and Kantere 1985; Weekman 1975; Volesky
arid Votruba 1992). When more phenomenolo~cal laws are included in the formulation of
a mathematical model this increases its extrapolation abifity but, unfortunately, also the amount of
computation power required for process simulation. Therefore, the final formulation of the model has
to be, according to Prater's principle (Weekmann 1975), a reasonable compromise between the cost of
computation and the value of approximation.
In bioengineering practice simplified deterministic models with lumped parameters are mostly
used (e.g, AJba et aL 1973; Pirt 1975; Biryukov and Kantere 1985; Takamatsu et al. 1981). The mass
conservation law serves as a phenomenological framework of the model. It is supposed that all para-
meters in the bioreactor have the same mean value. Tiffs is a very popular but rough approximation of
the real process. Anyway, for small bioreactors, when the medium is perfectly mixed and the physiology
of cells is limited by dissolved oxygen concentration, such a model is often recommended for scale-up
(e.g. Aiba et aL 1973; Kafarov et aL 1979; Singh et aL 1990).
A different situation occurs when the kinetic parameters depend on time (e.g. plasmid insta-
bility, age of cells, changes in apparent viscosity) or on the local position in the fermentor; e.g. for an
airlift fermentor in the riser and the downcomer sections of the bioreactor (e.g. Yonsel and Deckwer
1990). Here, we have to deal with the so-called distributed parameter models. In Table I the mass,
momentum and heat conservation laws are written in the general 3D-form of the distributed parameter
model. The effective diffusion coefficients in mass balances may be used either for modeling of mixing
pattern (e.g. Himmelblau and Bischoff 1968; Kafarov et aL 1975; Cirlin et aL 1986) or for diffusion in
microbial flocculates, pellets or film (e.g. Atkinson 1974; Roels and Kossen 1978; R o d s and Harder
1982; Roels 1982; BaiUey and Ollis 1987). The role of interface mass and heat transport coefficients
including changes of viscosity were discussed above.
A successful phenomenological model for bioreactor scale-up depends on the understanding
of the relationship between the microorganism and the environment (Mfilek et aL 1984); for the pro-
cess to be scaled up it is thus imperative to formulate a kinetic model that describes the physiology of
microbial growth, the rate of substrate consumption and the rate of product formation (Bazin and
Prosser 1988)o A detailed survey of simpler typical kinetic models can be found, e.g. in books (Pirt 1975;
Fredfickson and Tsuciffya 1977; Biryukov and Kantere 1985; Bailley and Ollis 1987). Recently we
summarized (Volesky and Votruba 1992) the most frequently used relationships for expressing simple
kinetics in simulation models (see Table II). Very often these simple kinetic terms are used in
combinations based on superimposition of several relationships between the different cellular
subsystems. Most often the sum or the multiplication product is used, depending on whether there are
sequential or parallel effects involved.
The problem of kinetic model identification and
estimation of its parameters based on experimental
Table H~ Summary of basickinetic terms used for data is like the procedure used in chemical reactor en-
development ofmathematicalmodels gineering (e.g. Himmelblau 1970; Hartmann and
Slinko 1972; Bard 1974; Votruba 1982; Kafarov et aL
Basic type Kinetic term 1982; Polak et aL 1984; Pogorelov 1988; Volesky and
of reaction rate Votruba 1992). The different time constants of the
particular processes may often cause a "stiff" behavior
R1 kc in the numerical solution of differential balances of
172 k Ca biomass, products and substrate by computer. In con-
R3 I, c / ( x + c)
Ra k Ca/(K + c ~) trast with experimental data obtained in chemical pro-
R5 tr | 1 - exp(-C/s cesses, which can be fitted by smooth curves, data
/is k exp(-C/K) from biological processes are rather scarce and are
R7 k K/(K + C) subject to large uncertainty caused by the biological
R8 k K/(K + C a) nature of the production strain. Application of linear
or nonlinear regression in which the sum of weighted
squares of residues is minimized (e.g. Himmelblau
1970; Bard 1974) may yield an erroneous estimate of model parameters in view of the linear model of
errors. The robust statistical approach (Hampel et al. 1986), especially the gnostic theory of measure-
ment (Kovanic 1984) which is based on the robust exponential model or errors makes it possible to set
up the algorithms that are effective for identification of complex kinetic models with lumped parame-
ters (Volesky and Votruba 1992).
1992 PHYSIOLOGICAL SIMILARITY A N D B I O R E A C T O R SCALE-UP - r e v i e w 341

The models with lumped parameters are mostly used for identification of microkinetics, based
on data obtained in laboratory fermentors where the concentration homogeneity is assured. When
using these biokinetic terms for the modelling of large scale unit, several methods are commonly used
for the description of spatial nonhomogeneities in a bioreactor under scale-up.
Himmelblau and Bischoff (1968), Ollis (1977), Kafarov et al. (1979) listed a number of exam-
ples based on the use of compartment models with a dead-water zone. Lately these models came into
extensive use (e.g. Khang and Levenspiel 1974; Andre et al. 1983; Bajpai and Reuss 1981; Oosterhuis
and Kossen 1983-1985; Crueger 1987) especially for the description of the hydrodynamics of
a multlphase flow in viscous media since the structure of the flows can be identified from the mea-
surements of the response of the bioreactor to a pulse of a tracer. Popovie e t al. (1983) used this
approach for modeling the gas residence time distribution in a large stirred tank bioreaetor.
As an illustrative example of this approach we can cite the scale-up study of polysaccharide
fermentation (Hubbart and Williams 1977, 1987; Margaritis and Zajic 1978) where the increased vis-
cosity of product qualitatively changed the flow pattern in the bioreactor. A similar approach was used
for scale-up and scale-down studies in fermentation of gluconic acid (Oosterhuis et al. 1985) and
industrial production of oxytetraeycline (Bosnjak et al. 1985). How this approach is efficient in the
development of a new process was demonstrated by Diversa Inc. (Hamburg) in the planning, design,
start-up and optimal control of large-scale cell plant culture (Ritterhaus et al. 1989, 1990) for 75 m 3
production scale bioreactor.
Another more complex and more accurate approach for a mathematical description of con-
centration nonhomogeneities in the bioreactor is the use of quasihomogeneous approximation o f the
flow pattern, especially in the scale-up of tubular systems where mixing nonidealities are described by
additional diffusion terms (see Table I). This form of mathematical expression of mass conservation
law is elegant, but not easy to handle in computer simulation. Anyway, it is recommended by many
authors as a school example for modeling and scale-up of nonconventional fermentors of the bubble
column or air-rift type, where great changes in hydrostatic pressure influence the solubility of oxygen
and carbon dioxide along the bioreactor (e.g. Kafarov et al. 1979; Yonsel and Deckwer 1990). On the
other hand, this approach is suitable for description of bioreactors with immobilized cells (e.g. Atkinson
1974; Volesky and Votruba 1992) because it makes it possible to take into account the effects of
external and internal transfer and axial mixing on the overall reaction rate.
The role of mathematical models with distributed parameters increases especially when
"a nonconventional fermentor" is designed.
The use of complex models, describing the physiology of cell in a bioreactor under scale-up is
complicated by considerable uncertainty, given by the biological nature of production microorganisms
and by the lack of detailed knowledge about cell physiology in new conditions. Often, such a situation
cannot be described by deterministic models summarized in Table I.
N o w , when using the computation power of supercomputers (Clementi et al. 1988) one can
solve the complex models of multiphase heterogeneous flow as predicted by Nigmatulin (1978), but the
problem remains how to deal with the uncertainty of the physiological response of a production strain.
One possible way is to use the mathematical apparatus of population balance (Randolph and
Larson 1971; Fredrickson et al. 1968). The advances in laser cytofluorometry make it possible to
determine the physiological state o f a population experimentally and establish what really happens with
cells during their individual life cycle. Image analysis and fractal geometry (Reichl et al. 1992) make it
possible to characterize and predict the changes in pellet morphology which is an important physiologi-
cal marker of production ability in cultivation of filamentous organisms.
The second possible way of treating the uncertainty in biokinetic terms of mass, heat and
momentum balances was suggested by Dorokchov and Kafarov (1989) where the deterministic models
with distributed parameters are combined with fuzzy formulation of boundary conditions and biokinetic
terms. Such models are successfully used in bioreactor control (Konstantinov and Yoshida 1989)o
According to our experience (~t&bfi~ek and Votruba 1992) such a combination of fuzzy and determin-
istic approach may be very helpful in bioprocess scale-up.
The principle of physiological similarity in bioreactor scale-up suggested here is independent
of whether physical or biochemical approaches are used. The synthesis of these two principles seems to
be the possible third way for bioreactor scale-up. Based on Rosen's idea (Rosen 1980) Gordejev and
Krumov (1988) formulated the procedure for column-type bioreactor scale-up as an optimization
problem under constrains. The constrains at the top level of system decomposition are accompanied by
the macrokinetics of the process; they are represented by the effectivity of hydrodynamic, heat and
mass transfer processes. The constrains at the "lower level" are coupled directly to physiological func-
342 $. VOTKUBA and M. SOBOTKA Vol. 37

lions describing the rates of growth, product formation and substrate utilization. This methodology of
scale-up was used for designing the industrial bioreactor of 1 000 m3 working volume.

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