FERMENTATION: CRITICAL PROCESS PHENOMENA AND

NEW TECHNOLOGY DEVELOPMENTS THAT AFFECT

YIELD AND PRODUCTIVITY

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BY PRISCILLA KAUFMAN, PAUL KUBERA AND TOM POST

TO ESTABLISH A FOUNDATION FOR UNDERSTANDING RADICALLY NEW FERMENTATION PROCESS TECHNOLOGY,

THIS ARTICLE REVIEWS THE BASIC MECHANICAL AND PROCESS PARAMETERS THAT DETERMINE FERMENTATION

TN YIELD AND PRODUCTIVITY. AN OVERVIEW OF THE OPTIONS AVAILABLE TO AND EMPLOYED BY

PHARMACEUTICAL MANUFACTURERS IS PRESENTED IN RELATION TO THESE VARIOUS PARAMETERS. FINALLY, NEW
PROCESS TECHNOLOGY EMPLOYING UP-PUMP~NG HYDROFOIL IMPELLERS IS INTRODUCED. THIS TECHNOLOGY
TAKES AN ENTIRELY NEW APPROACH TO FERMENTATION THAT IS RESULTING IN SUBSTANTIAL IMPROVEMENTS IN
YIELD AND PRODUCTIVITY.

Introduction
Aerobic fermentation is a big-chemical process used
throughout the pharmaceutical and biotechnology community
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to create the active ingredients for numerous compounds.
Micro-organisms are fed nutrients and oxygen which enables
them to reproduce and to metabolize those nutrients into other
by-products. Either the organisms themselves, their
components or their by-products are then harvested through
other down stream processes to create drugs such as
penicillin, Factor 8, hypolipidemic enzymes or other
intermediate pharmaceutical products.
Because of re-validation and capital investment concerns,
many phannacoutical manufacturers use fermenters installed
in the 1950s. TraD/Tionally, these fermenters use sparge rings
and multiple radial flow impellers known as Rushton turbines
to create conD/Tions that enable fermentation. However,
recent advances in process technology have occurred.
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levels are required when oxygen transfer is the limiting step.
In this decade, tanks have increasingly been retrofitted to
extend power per unit volume to increase dissolved oxygen
levels (especially in the critical phase) to improve yields.
To accommodate these volumes, mechanically agitated
fermenters typically have a tank height to diameter ratio (Z/T)
ranging from 2:1 up to 4:1, whereas air lift fermenters can
range up to 10:1. In full scale fermenters, that ratio is deter-
mined more by US traffic regulations, which limit the maxi-
mum tank diameter that can be transported on US highways,
than by particular mixing parameters. Uniformity as well as a
well mixed tank are much easier to achieve in more squat
shaped-tanks. Tall tanks, such as those usually used in
fermentation, tend to create individual mixing zones within
the tank that sigruficantly reduces blending.
Because of tank geometry, fermenters usually employ three or
more impellers of similar diameter to achieve proper mixing.

Regardless of whether a company needs to increase capacity or
simply replace or retrofit an older system, recent
improvements in mixing technology may enable
manufacturers to substantially increase yields as well as
improve productivity and product uniformity.
Mechanical Structure and the Impact on
Fermentation Results
Full scale fermentation tanks typically range from 35,000 to
175,000 liters (9,000 - 46,000 gallons) and average about
75,000 liters (20,000 gallons). The agitators themselves range
from 100 to 600 HP (75 - 450 kW), while most fall between
150 to 300HP(100 - 225 kW). High power per unit volume
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This number will vary; however, with the tank height to
diameter ratio (HIT). Shorter tanks may have less impellers
while taller tanks require more. Moreover, the spacing of
impellers is critical to the process results. When impeller
spacing is too far apart, dead zones occur. These dead zones
contribute to the starvation of the micro-organisms which in
turn leads to a reduction in yield and productivity.
To impart the power required for mixing, impellers with a
smaller impeller to tank diameter ratio (D/T) need to spin
faster relative to impellers with larger D/T ratios. Increases in
impeller speed; however, increase fluid shear within the tank
and that shear can be detrimental to the micro-organisms in the
broth. Most often the D/T ratios range from 0.3 to 0.5. For
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2 PHARMACEUTICAL ENGINEERING
“THE PLACEMENT OF BAFFLES IS NOT TRIVIAL. POSITIONING AND DESIGN OF BAFFLES ALONG WITH
THE PLACEMENT, TYPE AND DESIGN OF HEATING COILS HAS A SIGNIFICANT IMPACT ON THE FLOW
PATTERNS AND, THUS, ON THE OVERALL PROCESS RESULTS."
bacterial and yeast fermentations which are not particularly
shear sensitive, optimum D/T levels may be smaller, while
mycelial and, in particular, mammalian cell cultures, require
large D/T because of their shear sensitivity.
From an economic perspective, smaller D/T impellers have
fewer wetted surfaces and require less torque. This results in a
less expensive mixing system. On the other hand, large D/T
impellers require more torque and possess larger, heavier
wetted parts. Therefore, larger D/T impellers will cost more.
The cost of the impeller mixing system must therefore be
weighed against the value of the yield. Often the value of the
resulting yield improvements significantly outweighs the in-
creased cost of larger D/T impellers.
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Traditionally, gas (in the form of air or oxygen enriched
air) is introduced into the system through a sparge ring or
open pipe that is placed beloved the bottom impeller. In most
full scale fermenters, tanks are so tall that the hydrostatic head
of the liquid above the sparge rings requires the use of a
compressor to force air to the bottom of the tank. This has a
large impact on operating costs.
Recent research has shown that splitting the gas flow to
different locations within the tank can be beneficial to the
process. Placing the sparge ring higher up in the tank also
reduces hydrostatic head. This in turn allows for more cost
efficient air blowers rather than compressors. For large fer-
mentation plants, this results in a significant reduction in
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annual operating costs.
Heat transfer surfaces are placed between the impeller blade
tips and the tank wall to regulate the temperature of the broth.
These can be helical coils, vertical plates, or vertical coil
bundles - all of which have an impact on the flow pattern
developed in the fermenter.
It is important that agitators supply high flow over these heat
transfer surfaces to minimize hot and cold spots which will
have an adverse effect on temperature sensitive fermentations.
In traditional systems, it is not unusual to see temperature
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Figure 1. Baffled radial-flow turbine pattern.
SEPTEMBER/OCTOBER 1997
differences within the tank greater than +3°C. Improving
mixing has been demonstrated to decrease temperature gradient
to less than 0.2°C in industrial fermenters.
Baffles are very important in the majority of fermentation
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processes. Baffles reduce the inefficient tangential velocity
component of all impellers to produce a more efficient radial
or axial flow. The placement of baffles is not trivial.
Positioning end design ofbaines along with the placement,
type end design of heating coils has a significant impact on
the flow patterns and, thus, on the overall process results.
Process Requirements
There are several concurrent mixing objectives in fermenta-
tion including
Solids Suspension: to suspend the media and biomass.
Blending to uniformly blend the broth containing the media,
micro-orgarusms, nutrients and any reagents as well as to
maintain temperature and pH constancy throughout the broth.
Blending is very dependent upon the mechanical arrangement
of the fermenter as outlined above.
Dispersion: to disperse the gas uniformly throughout the
broth (and thereby increase gas hold-up), reduce the size ofthe
gas bubbles and prevent flooding.
Mass Transfer: to dissolve the oxygen into the broth (gas-
liquid mass transfer) enabling the dissolved oxygen to pass
through the ceil walls of the micro-organisms as well as
enable carbon dioxide flux in the opposite direction as the
microorganisms metabolize (liquid-solid mass transfer).
Heat Transfer: to facilitate the removal of heat created as a
result of cell metabolism as well as the heat generated through
the dissipation of mixing energy via viscous shear.
These basic mixing principles have been applied to fermenta-
tions in the pharmaceutical market since the 1960s. Research
on fermentation mixing technology tends to focus on mass
transfer limitations because it has been assumed that mass
transfer is usually the rate limiting factor in fermentation.
Fundamental research on fermentation has always been done
on the small scale where mass transfer has indeed been the
rate limiting factor in the process.
Because blending is considered to be less complex than
mass transfer, it is the most overlooked parameter of full-scale
fermentations. None-the-less, blending efficiency has a sig-
nificant impact on yield and productivity. In fact, if the time
scale of mixing approaches the time scale of mass transfer, the
rate limiting factor shifts to blending. In full-scale fermenta-
tion, blending must be considered to maximize yield and
productivity.
All energy that is applied to a fluid mixer eventually appears
as heat through viscous shear. Ultimately, energy is dissipated
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SEPTEMBER/OCTOBER 1997
Figure 2. Time independent shear rate.
dissipated at the micro-scale level where it is transformed to
heat as a result of viscous shear. In the case of fermentations
of long duration, this heat is significant and must therefore be
removed through heat transfer surfaces. Otherwise, the heat
build up can be detrimental to the micro-organisms.
as well as productivity gains.
Shear Rates
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Recent improvements in mixing technology discussed later
in this article affect blending, dispersion, heat transfer and
mass transfer as well as the interaction between each of these
processes and are resulting in significant yield improvements
Most common batch fermentations are bacterial, mycelial or
yeast based broths, and while all are affected by shear, myce-
lial fermentations are by far the most shear sensitive. More-
over, scale-up development of mammalian cell culture and
rDNA is limited by shear sensitivity. To understand how
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shear can affect fermentations, average and maximum shear
must be understood. This is especially important for shear
sensitive fermentations such as newer cell cultures where
processes are moving from pilot to production scale
operations. This is because in conventional fermentation
systems, while average shear rates decrease on scale-up,
maximum shear rates increase, and that can have a
significant impact on the process results.
Time independent fluid shear rate is defined by a velocity
gradient which is the change in average velocity over a given
distance. Time dependent fluid shear rate is defined by
instantaneous velocity gradients over a given distance.
Usually, these are determined in the impeller zone as well as
throughout the entire batch. These data are usually obtained
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with hot film aenomometry, a Laser Doppler Velocimeter or
with a Particle Image Velocimetry.
When looking at time indeperident shear, as described by
average velocities, actual velocity fluctuations are obscured,
but it is this variability in velocities that can have a significant
impact on shear sensitive fermentations. Therefore, to account
for this, velocity fluctuations also must be accounted for by
calculating the root mean square (RMS) value of the fluctua-
tions (obtained by squaring the fluctuations, averaging them
and then taking the square root). This is a useful representa-
tion of time dependent shear.
Shear rates operate throughout the tank at different mag-
nitudes and scales. The shear rate determined by the average
velocities affects particles in the order of several hundred
microns. The fluctuating velocity shear rates deal with micro
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PHARMACEUTICAL ENGINEERING 3
scale processes and affect particle sizes or cluster sizes of
200 microns or less. It is on the micro-scale level that both
mass transfer and diffusion occur.
The highest shear rates are in the impeller zone, generated by
the discharge velocity. This discharge velocity can be char-
acterized by an average impeller shear rate and a maximum
impeller shear rate. Regardless of impeller type, the average
impeller shear rate is only a function of the impeller speed.
Maximum shear rates, on the other hand, depend upon the
type of flow pattern generated. For radial flow impellers like
the Rushton Carbine, the maximum shear rate is a function of
impeller speed and diameter or tip speed. For axial flow
ungellers; however, the maximum impeller shear rate is also
only a function of speed.
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Upon scale-up, impeller speeds are always less than those
for pilot scale equipment. Therefore, average shear is always
less, whereas maximum shear rates always increase for radial
impellers, but decrease for axial flow impellers. Thus, for full-
scale shear sensitive fermentations, it is not unusual to see
better yields when using axial flow impellers.
Coalescence
Coalescence is the characteristic of substances coming to-
gether. In fermentation, this describes the tendency for smaller
bubbles to converge with other bubbles to form larger bubbles.
Most all pharmaceutical fermentation processes represent
coalesce systems.
In studying aerobic fermentation processes, the gas-liquid
behavior of these processes is traditionally evaluated using
air-water models. While theoretically these models do represent
coalescing systems, in reality they do not effectively behave so.
Once the gas is dispersed in water, it does not rapidly coalesce
to create the "bubbling" and "surging" seen in real
fermentations. Moreover, salts and electrolytes are often added
to the fermentation broths as part of the testing process, further
causing the medium to behave more like a non-coalescing
system. This has implications for the traditional way
fermentation processes have been analyzed and for the actual
effect of shear on full scale fermentation broths.
For dispersion processes where bubbles do not tend to
coalesce, the effect of the average shear rates and maximum
shear rates on bubble size would determine the ultimate
dispersion. Therefore, if the process lasted long enough, arid
most fermentation processes do last several days, the ultimate
bubble size would he determined by the maximum shear rate
around the impeller since all bubbles would eventually move
through this zone.
This suggests that in a water-air model which acts effectively
as a non-coalescing system, the ultimate air bubble size would
be determined by the maximum shear rate around the impeller.
This is an inaccurate representation of most fermentations
which have higher viscosities.
Because most fermentation processes are coalescing sys-
tems, they cannot be adequately understood by air-water
models which more reflect non-coalescing systems. In reality,
fermentations broths are of significantly higher viscosity than
is represented by water. Moreover, as the micro-organisms
within a fermentation broth multiply, viscosity increases.
Viscosity acts as a drag inducing action such that the
reduction or lack of shear, enables bubbles to increase in size
(ie to coalesce). In aerobic fermentations; therefore, air
bubbles will tend to coalesce, and there is an equilibrium
between dispersion and coalescence. In this case, the entire
spectrum of shear rates within the tank become relevant.
Because of the pressure differentials around an operating
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4 PHARMACEUTICAL ENGINEERING SEPTEMBER/OCTOBER 1997

impeller, air bubbles tend to migrate toward areas of lower

pressure and coalesce. In aerobic fermentations, large bubbles
can get caught behind the impeller blades. This is especially
pronounced in Rushton Turbines, but is a problem with all
down-pumping impellers as well. Newer technology discussed
below follows the natural flow patterns of coalescing
mediums. This eliminates the opportunity for bubbles to
aggregate into larger bubbles and stall the impeller.
Flooding occurs when the gas that is pumped into the
fermentation broth prevents the impeller from mixing the
broth. There is a propensity for all radial flow and down-
pumping axial impellers to flood in gassed conditions. To
counter the possibility of flooding, the impeller diameters
must be sufficiently large (which traps air bubbles) or the
speed at which they rotate must be sufficiently great (which
“ALL AXIAL FLOW IMPELLER TECHNOLOGY
IN FERMENTATION AS WELL AS IN MOST ALL
PROCESSES IS PREFACED ON THE CONCEPT
OF DOWN-PUMPING.”
high efficiency axial flow impellers. This allows for improved
blending which, depending upon the process, in turn leads to
yield improvements. Because axial flow impellers have rela-
tively lower power numbers than radial flow impellers,
significant reductions in energy costs also are realized.

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increases average shear). Fluctuations in velocity increase,
and micro-level shear rises which can have a detrimental
effect on shear sensitive cultures.

Impeller Technology
Traditionally, Rushton-type radial flow impellers have been
the impellers of choice in fermentations because they are
considered to be good for gas dispersion. However, these
turbines have relatively high power numbers (they require

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more power than other impellers of comparable diameter at a
given speed) and exhibit a significant decrease in power draw
upon aeration. Because the mass transfer coefficient is depen-
dent upon aeration rate and power per unit volume, this
implies a reduced mass transfer potential.1
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Figure 4. Fermenter mixer employing A310 axial flow upper

Figure 3. Rushton turbine. impellers.

Moreover, while the purpose of the bottom radial flow In the late 1980s, further improvements in the yields of
aerobic fermentation were realized with the introduction of
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impeller has been to disperse gas as it enters the tank and to

decrease bubble size to increase the surface area of the gas-
liquid interface, the upper impellers do not further decrease the
size of these bubbles (in non-coalescing fermentations). Rather,
their function is to maintain the dispersion and circulation in
the upper part of the vessel. Finally, compartmentalization of
the flow patterns is a problem with multiple radial flow
impellers like Rushton turbines. This results in increased ring
time as well as poor distribution of nutrients and dissolved
oxygen.
In the early 1980s, it was recognized that the purpose of the
upper impellers was primarily to provide uniform blending and
to allow contact between the air, broth and nutrients so that
mass transfer can occur. Because the upper impellers do not
contribute to air dispersion, they can often be replaced with
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high solidity ratio hydrofoil impellers. These impellers have a
high ratio of total blade area to total projected sweep of the
impeller. With large, profiled and angled blades, these impel-
lers produce strong axial flow patterns while providing supe-
rior gas dispersion. When high efficiency axial flow impellers
cannot effectively handle gas, high solidity ratio hydrofoil
impellers offer an effective alternative. Because oxygen mass
transfer efficiency is improved, yield improvements are real-
ized.
New Process Technology
All axial flow impeller technology in fermentation (as well as
in most all processes) is prefaced on the concept of down-
pumping. The impellers all circulate the fermentation broth in
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SEPTEMBER/OCTOBER 1997
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Figure 5. A315 impeller.
a downwards motion with the intent to hold the gas down and
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longer in the tank. Therefore, in aerobic fermentation, the
impeller pumps against the flow of the rising air. However,
recent research suggests that using hydrofoil impellers in an
up-pumping mode can increase mass transfer, improve blend-
ing and heat transfer and decrease blend time. This results in
decreased reaction times, improved nutrient utilization, im-
proved product uniformity and purity and decreased mechani-
cal vibration. In the retrofit of actual systems, yield increases
of 20 to 40 percent have been obtained.
Several factors likely contribute to such phenomenal im-
provements in yield. In the case of airlifts, up-pumping en-
hances the natural flow pattern of the gassed system. In
mechanically agitated fermenters, up-pumping impellers are
not working against the gas stream, and in fact, they cannot
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flood.
Unlike Rushton turbines which have much higher power
numbers and which exhibit a large drop off in horsepower
upon aeration, up-pumping axial flow impellers do not
demonstrate a significant difference between gassed and
un-gassed horsepower. As stated earlier, the mass transfer
coefficient is dependent upon aeration rate and power per unit
volume. This therefore suggests improved mass transfer
potential at equal un-gassed agitator power.
Recent studies using up-pumping technology also demon-
strate improved heat transfer and temperature uniformity.
Temperature gradients throughout the entire tank were found
to be less than +0.2°C. This is a significant improvement over
the temperature distribution seen in traditional radial flow
impeller systems in which flow compartmentalization is often
a problem.
PHARMACEUTICAL ENGINEERING 5
“EVIDENCE SUGGESTS THAT THE MAXIMUM SHEAR
RATE DOES NOT CHANGE SIGNIFICANTLY ON
SCALE-UP, AND THIS HAS MAJOR IMPLICATIONS
FOR ALL SHEAR SENSITIVE FERMENTATIONS.”
Full scale up-pumping systems also demonstrate a four-fold
decrease in blend time. The dramatically improved blending of
nutrients and reagents can contribute to yield improvement as
well.
By encouraging air bubbles to circulate in the direction
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which they are naturally prone to move, up-pumping hydrofoil
impellers do not provide a surface for bubbles to coalesce.
Bubbles cannot collect under the impellers. Bursting bubbles
can cause cell damage in mycelial broth. By improving disper-
sion and reducing coalescence, "bursting" and "surging" is
decreased, suggesting a more hospitable environment for
mycelial culture.
Finally, as stated earlier, in the scale-up of traditional
fermentation systems, the maximum shear rate increases while
the average shear rate decreases. Maximum shear rates of
hydrofoil impellers do not increase on scale-up. With up-
pumping systems, evidence suggests that the maximum shear
rate does not change significantly on scale-up, and this has
major implications for all shear sensitive fermentations.
Conclusion
Each fermentation process presents unique constraints whether
they be from the process result (the product), the process itself,
validation concerns or capital expenditure. An understanding
of basic fermentation mixing principles and aerobic fermenta-
tion behavior will help manufacturers determine whether up-
pumping hydrofoil impellers, high efficiency axial flow impel-
lers or traditional radial flow impellers meet their specific
objectives.
To do so, impeller characteristics such as average shear and
fluctuating velocity as well as environmental parameters such
as viscosity and coalescence must be considered. This ensures
that the fermentation system is properly designed to maximize
process performance.
As pharmaceutical companies continue to come under price
pressure in the 1990s, improvements in manufacturing that
result in operating profit growth will be critical to a company's
success. The potential for significant yield improvement
through the use of up-pumping hydrofoil impellers in aerobic
fermentation is one means for manufacturers to improve their
bottom lines.
References
1. Caroline M. McFarlane, Alvin W. Nienow, "Studies of High
Solidity Ratio Hydrofoil Impellers for Aerated Bioreactors. 1.
Review," Biotechnology Progress 1995, 11, 601-607.
2. Klaas van't Riet, Johannes Tramper, Basic Bioreactor Design,
New York, New York, Marcel Dekker, Inc. 1991.
3. James Oldshue, Fluid Mixing Technology, New York, New
York, Chemical Engineering, McGraw-Hill Publications Co.
1983.
4. N. Kioukia, A.W. Nienow, N. Al-Rubeai, A.N. Emery, "Influ-
ence of Agitation and Sparging on the Growth Rate and
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6 PHARMACEUTICAL ENGINEERING
Infection of Inset Cells in Bioreactors and a Comparison
with Hybridome Culture,” Biotechnology Progress 1996,
12, 779-785.
5. Kenneth S. Lally, James Y. Oldshue, Ronald J. Weetman,
"Mass Transfer and Fluid Mixing in Fermentation," Pre-
sented at American Institute of Chemical Engineers, No-
vember 12, 1985 Annual Meeting, Chicago, Illinois.
6. James Oldshue, "Mixing in the Fermentation Process,"
Annual Reports of Fermentation Processes, Vol 6 1983,
75-99.
N
7. J. Y. Oldshue, C.K. Chan, H. Zemke, K. Bruegger, "Fluid
Mixing Variables in the Optimization of Fermentation
Production," Process Biochemistry Vol 13, No 11, 1978.
8. J. Y. Oldshue, "Some Scale-up Considerations for Fermen-
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tation Mixers," Advances in Bio-Technology, Vol 1,517
-522, Proceedings of the Sixth International Fermentation
Symposium, London Canada, July 20-25, 1980.
About the Authors
Priscilla Kaufman holds a BA from Wellesley College and an
MBA in operations and finance from Columbia University.
Before joining Lightnin, Kaufman worked in various interna-
tional commercial positions for Lederle Laboratories and later
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consulted for Fisons Pharmaceuticals and Bausch and Lomb.
Currently, Kaufman leads a biotechnology team of engineer-
……
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SEPTEMBER/OCTOBER 1997
ing, research and manufacturing experts that is responsible for
addressing Lightnin's activities within the pharmaceutical and
biotechnology industries. Kaufman is a member of ISPE.
Paul Kubera received both his BS and ME degrees in chemi-
cal engineering from Cornell University. Kubera began his
career as a research engineer with US Steel Corporation where
he specialized in environmental control, solids-liquid separa-
tion and by-products processing. Kubera joined Lightnin 17
years ago as a research engineer. Since then, he has held
various positions in engineering and research. His areas of
particular interest are gas-liquid mass transfer, biological
processes, waste and water applications and minerals process-
ing. Kubera is a member of the American Institute of
Chemical Engineers and the North American Mixing Forum.
Dr. Tom Post received his chemical engineering degrees from
the Swiss Federal Institute of Technology in Zurich, Switzer-
land where he completed his doctorate in 1983. From 1983-
1987, Post was an associate senior investigator for SmithKline
Beecham where he was responsible for the scale-up of many of
SKB's new organic synthesis projects as well as the improve-
ment of existing product lines. In 1987, Post joined Lightnin's
R&D group where he has focused on mixer process perfor-
mance. From 1990-1993, Post was the director of Lightnin's
German R&D facility in Munich, and in 1995 he was named
Vice President of Mixing Technology at Lightnin in
Rochester, New York. His most recent projects deal with
coalescing and non-coalescing gas liquid dispersions.
Lightnin, 135 Mount Read Blvd., Rochester, NY 14611.