RHEOLOGY OF FERMENTATION BROTH

The fermentation broth is a very complex soup or solution. Fundamentally, the fermentation broth
is the sea of nutrients in which the microorganisms grow, reproduce and 'swim' . The fermentation
broth supply the microorganisms with all the nutrients the microorganisms need to grow and
produce the various fermentation products.

The fermentation broth too act as the medium for various physical, biochemical and physical
reactions to take place. The fermentation broth will be implicated in all the mass and heat transfers
that occur within the fermentor, and it will be the medium that holds the fermentation products
formed.

The nature and composition of the fermentation broth temporally and spatially will affect the
efficiency of the fermentation process. The interactions between the fermentation broth and the
various components is complex and affect both directions

WHAT IS IN THE FERMENTATION BROTH?

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At any time the composition of the fermentation broth is complex consisting of anything that ends
up in the fermentation broth. This includes:

1 Raw substrates
2 Fermentation products
3 Microorganisms and its derivative components
4 Chemical additives added to the fermentor
5 Gases such as oxygen and other metabolic gases

All three main phases; solid, liquid and gases are present in the fermentation broth and their
possible interactions

RHEOLOGICAL PROPERTIES OF FERMENTATION BROTH

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One of the most important singular properties of the fermentation broth will be its rheological or
viscosity characteristics. Let us not stick to strictly to the definition and quantification of rheology
but rather try to appreciate it from its behaviour and its impact.

We always 'picture' fermentation broth as a thick gooey sticky mixture that is thick and viscous
compounded by rising bubbles of gas exploding at the broth surface. Maybe this picture is too
dramatic but in a way it is true!

The viscous nature or the rheological properties will affect the mixing regimes of the fermentor.
Viscosity is not a simple but a complex phenomena that is always changing and responding to
various parameters. Very rarely can we describe a fermentation broth as following a Newtonian
behaviour. In most cases it is a complex combinations of various Non Newtonian behaviour.

This poor understanding of the fluid behaviour of the fermentation broth will affect the efficiency
of mixing and liquid circulations resulting in poorly controlled or less economical fermentation
process

WHAT CAUSES THE BROTH TO BE VISCOUS?

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The viscousness of the fermentation broth is caused by the interactions of the various components
in the fermentation broth. The interactions may occur between the components of the broth and
the water or it could result from the interactions between the components themselves. Such
interactions in the viscosity of the broth could occur at the level of the ions and molecules which
involve the various ionic forces or it could involve at the macrolevel such as between the various
biopolymers tangling and sliding with each other. The overall result will be that the fermentation
broth will be viscous.

Now let us look at one of the components which make the fermentation broth viscous, that is the
contribution of sugars to the viscosity. Sugar or the carbohydrates are the main carbon source in
any fermentation media and supply the carbon needed for energy and skeleton structures of the
cells and organic compounds

Experience have shown to us that sugar in solution is sticky, but dry sugar is not sticky. The
stickiness or viscousness of the sugar in solution is caused by hydrogen bondings which develop
between the sugar molecules and water. During the interactions of sugar and water the hydrogens
in the water molecules and the hydrogen in the sugar molecules have an attraction for each other.
Thus it is the hydrogen bondings that make the sugar sticky!

Thus we see that most of the viscosity in the fermentation broth is caused by the various hydrogen
and other ionic bondings

IMPACT OF VISCOSITY ON FERMENTATION

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The most crucial effect of viscosity is that it makes the situation very difficult to achieve proper
and complete mixings. This will affect the various mass transfer processes that occur in the
fermentor. poor mixings due to high viscosity will also result in the formation of various physical
and chemical gradients

Viscosity makes scaling up studies difficult due to the change in behaviour of the fermentation
broth such as difficulty in mass heat transfers, solubility of components and gases and mixings at
the upper scale of fermentation process
Aeration
Bioreactors are widely used to cultivate cells during the development and manufacturing of
modern biopharmaceuticals. Cells are very sensitive to changes in the culture environmental
conditions, such as aeration, agitation, nutrients, and pH. This article discusses the importance of
aeration and available options to control the oxygen mass transfer coefficient (kLa) within a
bioreactor.
Delivering oxygen to cells
In cell culture, oxygen is a key substrate for growth, production, and maintenance activities. Cells
obtain their oxygen in free and noncompound forms, called dissolved oxygen (DO). One of the
most important functions of bioreactors is providing dissolved oxygen to cells continuously
through a process called aeration.
Aeration in the bioreactor typically occurs when:
1. Oxygen diffuses through overlay to the cell culture medium interface.
2. Oxygen from the spargers dissolves in the cell culture through convection with the help of
agitation.
Agitation disperses the oxygen bubbles and promotes mass transfer of the gas bubbles through the
gas-liquid (cell culture medium) interface. The rate of oxygen transfer (OTR) from gas to liquid
interface is a function of physicochemical properties of the cell culture medium, the geometrical
parameters of the bioreactor, and presence of cells.

Diagram of a gas bubble in liquid, showing how the bubble is released, solubilized, and
transferred to a cell.
Oxygen utilization rate (OUR) is often cell line-dependent. The following table lists the rates for
common industrial cell lines (Ruffieux, P. A. et al. and Xiu, Z. L. et al.)
Oxygen utilization rates of cell lines typically used in biomanufacturing

Cell line OUR [10-13mol/cell/h]

DG44 (CHO) 2

CHO 5.0–8.04

NSO (myeloma) 2.19–4.06

MAK (hybridoma) 4.16

FS-4 (human diploid cells) 0.5

HFN7.1 (hybridoma) 2

Due to its low solubility in liquid phase and increasing metabolic consumption by the cells with
time, oxygen is supplied continuously to the cell culture. Oxygen supply is carefully controlled for
optimal cell growth by manipulating bioreactor parameters.
During batch cell culture, OUR (or OTR) is initially low during the lag phase, where cells are
self-synthesizing and there is little gain of cell density. As cell density increases during the
exponential phase, OUR increases until OTR becomes a limiting rate, as determined by the mass
transfer of oxygen into the bulk liquid.

Phases of cell growth

The OTR and OUR rates are correlated by the oxygen mass transfer coefficient, kLa. Therefore,
the OTR, through its correlation to kLa, defines a theoretical maximum cell density that could be
achieved in cell culture.
Utility of kLa values
Because of this association with cell density, kLa values are particularly useful in the following
scenarios:
Scenario 1: Evaluating scalability within the same bioreactor platform
The conventional scale-up of bioprocesses is based on physicochemical and geometric similarity.
kLa is kept constant for this scenario. The OTR should remain constant for a bioreactor platform
with geometric similarity (such as Xcellerex bioreactors). Bioreactor physical characteristics at the
different scales are altered to provide the necessary OTR at controlled temperature, pH, and DO to
achieve the target cell density.
Scenario 2: Technical transfer across different bioreactor designs
During the comparison, kLa is utilized as a target performance metric when a process is
transferred from one bioreactor platform to another design. Bioreactor hardware design (e.g.,
stirrer geometry and aeration-sparger option) and running parameters (e.g., gas flow rate or power
input) are altered to achieve a similar kLa, providing a similar cell density.
Equation for kLa
Imagine a gas bubble in liquid. For this discussion the gas bubble contains oxygen, and the liquid
is the liquid in a bioreactor. kLa can be represented by the following equation:
kLa = kL × a

Where kLa is the mass transfer coefficient from the gas to liquid phase, given in sec -1

kL = liquid side mass transfer coefficient (resistance in gas side film can be neglected)

a = bubble surface (available for diffusion)
Phases of cell growth.
Key variables that impact kLa values
Any change to process and engineering parameters or to physical characteristics will have an
impact on kLa and should be considered when evaluating bioreactor platforms and performing
scaling calculations.
Here are four key variables that can affect kLa values:
1. Gas bubble size
When gas bubble size decreases, surface area and gas residency time increases, causing bubbles to
stay in the culture longer. Thus, there is a greater opportunity for oxygen to release mass transfer
into the cell culture medium. An increase in this oxygen residence time improves kLa.
2. Mixing
In a bioreactor, mixing is used to eliminate gradients of concentration (cell, gas, medium, and
nutrient), temperature, and other properties. Mixing time is widely used to characterize mixing
efficiency in a bioreactor. Mixing efficiency is one of the most significant factors affecting both
performance and scale-up in a bioreactor.
Gas bubble size and residency time are highly dependent upon three mixing conditions: impeller
type, speed, and location(s). kLa values generally increase as tip speed increases. However, tip
speed is proportional to shear forces that can lead to cell death. Bioreactors, therefore, are
designed with different impeller types, combinations, and locations to achieve target kLa values
without creating these shear forces.
Generally, kLa values are closely associated with impeller design, with Rushton typically higher
than paddle, which is typically higher than marine and pitched impeller.
Diagram of a bioreactor process showing key factors that can influence kLa values.
3. Air flow rate
Higher oxygen availability drives kLa increases. Increasing oxygen supply to a bioreactor drives
this availability and can be controlled by modifying concentration (air vs O 2 enrichment) and
volumetric flow. Although high kLa values are desirable, it is important to consider the actual
operating conditions and implications to cell viability and associated process costs.
For example, high air flow rates can cause cell damage due to shear forces. Excessive foam might
also be generated, requiring a high concentration of antifoam that could hinder downstream
processing. Additionally, higher air flow rates require a larger exhaust filter area, driving
consumable cost increases.
4. Properties of the liquid or medium
During cell culture, small bubbles collide and coalesce to form larger bubbles, decreasing surface
area (a) and subsequently kLa. Be aware of reported kLa values in which high salt concentrations
are used, because this can prevent bubble coalescing. Antifoaming agents are used to influence
surface tension, resulting in reduced bubble coalescence and foaming.
However, this principle does not always lead to increases in OTR wherein antifoam also reduces
bubble mobility, which subsequently reduces the kLa (Doran, P.).
Other factors that affect cell culture kLa
5. Measurement method
Several different methods are used. Most commonly the nitrogen stripping (i.e., gassing-out)
method is employed.
When scaling a process within the same platform, it is important to use an identical method for
measuring kLa. kLa, when combined with process engineering parameters (i.e., tip speed, power
input), can be used to experimentally determine the cell density in a larger bioreactor compared
with a smaller bioreactor.
6. Temperature
Increasing temperatures inversely affects both the volumetric mass transfer coefficient and oxygen
solubility in culture medium. Oxygen solubility in pure water falls with increasing temperature
(i.e., -0.5 × 10-3 kg/m-3 between 35°C and 30°C; Doran, P.).
Therefore, it is important to note the temperature conditions from vendor-supplied characterization
data.
7. Sparger characteristics
kLa values will vary widely with sparger characteristics, including number, pore size, and surface
area, because these factors affect bubble size, gas velocity, and flow rates.
Conclusion
As discussed here, kLa is impacted by multiple factors. A thorough understanding of a bioreactor
platform’s physical design, mixing mechanism, sparging options, as well as cell line
characteristics will inform decision-making when scaling cell culture processes up and out.
Agitation
Various types of oxygenation hardware designs have been developed to suit specific purposes in
bio-systems.
For example, oxygenation required in cell culture uses bioreactor hardware of various designs to
take care of bio-system characteristics.
For this purpose, conventional stirred tank bioreactor (STBR) designs have been used for a long
time to oxygenate or aerate the bio-reaction liquid.
Many bacteria and yeasts, as well as a few other microorganisms, can grow well when floating
free in a liquid culture medium in vessels with a capacity as high as several thousand liters,
resisting damage even when they have proliferated to form a thick suspension, and even when the
suspension is oxygenated and agitated vigorously with a mechanical sparger and agitator.
Many microbial, mammalian, and plant cells are different. They are either-highly shear sensitive,
flocculating or pellet forming, or larger than most microorganisms, more fragile and more
complex. Moreover, the delicate plasma membrane that encloses mammalian cells is not encased
in a tough cell wall, similar to many plant cells. Mammalian and plant cells that multiply in
suspension can usually be cultivated by oxic techniques, similar to those used in microbial
fermentation.
However, most animal or mammalian cells do not grow at all in suspension, but grow only when
they can attach themselves to a surface. They are called anchorage-dependent mammalian cells.
Anchorage-dependent cells have traditionally been grown on a large scale on the inner surface of a
roller bottle reactor. As the bottles roll, the cells are exposed to oxygen in the air space and to the
growth medium. The design of rotary oxygenators has been used also in microbial bioprocessing
for product formations as well as in effluent treatment for pollution control (rotary biological
contactors).
In the human body, the need for oxygenation requires no emphasis. With respect to oxygenation,
apparently the function of an artificial lung is to oxygenate at least 4.5 ml of O 2/100 ml of blood at
the maximum rated blood flow. Many manufacturers design and produce membrane oxygenators
that utilize micro-porous polypropylene membrane for gas exchange. Designs of these
oxygenators include submerged bioreactor oxygenator, hollow fibers, a flat-plate type, or
sometimes other types of configurations. Hollow-fiber membrane oxygenator designs have been
greatly improved in the past few years.
In the perfusion mode of the mammalian cell culture system, the use of tubular spiral film oxygen-
permeable membrane oxygenator has become a common technique. This type of cell culture
technique using perfusion oxygenation has been developed mostly to avoid fluid mechanical
damage of animal cells in conventional oxygenated bioreactors.
In the plant cell culture system, the oxygenation situation may become very critical in terms of
cell growth and cell division. Whatever the oxygenator used, the biomass yield usually serves as a
valuable parameter in bioprocessing. For prediction of biomass yield, oxygen efficiency (η 0) has
been used by some investigators. Minkevich and Eroshin proposed oxygen efficiency by defining
it as the ratio of the amount of electrons conserved in biomass over the amount of electrons
available in organic substrate by aerobic combustion to HCO3–.
t has been shown that η 0 follows from YDX (biomass yield on electron donor, per mol, or per C-mol
for carbon compounds (C-mol/mol) using only the black box conservation relationship. Hence,
η0 being a true black box, it can only be applied to aerobic systems and, therefore, lacks general
applicability. A simple example of black box information for aerobic growth of Pseudomonas
oxalaticus on oxalate with a yield of 0.586 C-mol/mol, as given above, provides the corresponding
macro-chemical mass balance relationship as follows:
– 5.815 C2O42- – 0.2 NH4+ – 1.857 O2 – 0.8 H+ – 5.414 H2O + 1.0 CH1.5 O5 N0.2 + 10.63 HCO3– = O
Here, there is no intrinsic limit based on the second law of thermodynamics.