Biochem Docu

PAMANTASAN NG LUNGSOD NG MAYNILA
University of the City of Manila

College of Engineering and Technology
Department of Chemical Engineering
Biochemical Engineering:
DESIGN AND ANALYSIS

OF BIOREACTORS
Submitted by:
DIONISIO, Charles Amiel P.
MACASIL, Renzo C.
BSChE V-I
Submitted to:
Dr. Denvert C. Pangayao
Fluidization and Dust & Mist Collection
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Fluidization and Dust & Mdsddsdsist Collection
Bioreactor Analysis and Design

To design a bioreactor, some objectives have to be defined. The decisions
made in the design of the bioreactor might have a significant impact on overall
process performance. Knowledge of reaction kinetics is essential for
understanding how a biological reactor works. Other areas of bioprocess
engineering such as mass and energy balances, mixing, mass transfer and heat
transfer are also required.
A fermentor (bioreactor) is a closed vessel with adequate arrangement for
aeration, agitation, temperature and pH control, and drain or overflow vent to
remove the waste biomass of cultured microorganisms along-with their products.
A fermentor is used for commercial production in fermentation industries
and is a device in which a substrate of low value is utilized by living cells or
enzymes to generate a product of higher value. Fermentors are extensively used
for food processing, fermentation, waste treatment, etc.
General Design Guide

STIRRED TANK BIOREACTORS
The most important bioreactor for industrial application is the conventional
mixing vessel, which has the dual advantages of low capital and low operating
costs. Figure 3 is a schematic diagram for such a reactor. Vessels for laboratory
experiments of volume up to 20 liters are made of glass. For larger volumes,
construction is made of stainless steel. The height: diameter ratio of the vessel can
2
vary between 2:1 and 6:1, depending largely on the amount of the heat to be
removed, and the stirrer may be top- or bottom driven. All tanks are fitted with
baffles, which prevent a large central vortex being formed as well as improve
mixing. Four baffles are used for vessels less than 3 meters in diameter, and six to
eight baffles are used in larger vessels. The width of the baffle is usually between
T/10 and T/12, in which T is the tank diameter.
Height of vessel to diameter:
3
Diameter of vessel to baffle:
Impellers: The ratio of the impeller diameter to the diameter of the tank
(di/dt) should be between 0.3 and 0.5. In the case of using radial flow impellers
the ratio should be approximately 0.3. If the impellers are too small they will not
generate enough fluid movement, whereas if they are too large they require
much more power and become less efficient. Typically stirred tank fermentors
employ Rushton turbines using either a single impeller or a set of impellers for tank
mixing. Recent developments in impeller design have led to the use of several
different types of impellers (e.g. Smith, He3, A320, Intermig). Even though these
new types of impellers claim to produce better mixing and have less power
consumption, typical fermentors only employ standard Rushton turbines.
Impeller Spacing: The spacing between impellers should be 1.0di to 2.0di,
where di is the diameter of the impeller. In addition, the bottom-most impeller
should be located 1.0di from the bottom of the tank. If the impellers are spaced
too close together (less than 1.0di) the power imparted to the fluid can get as low
as 80% of that obtained from proper spacing. On the other hand, if the impellers
are spaced too far apart the fluid does not experience adequate mixing. Thus,
the number of impellers can be determined from the following equation:
where HL is the height of liquid in the vessel and ni is the number of impellers
However, this is assuming all the impellers are spaced equally between the
bottom of the tank and the liquid surface. As stated before, the bottom-most
4
impeller is usually spaced one impeller diameter from the tank bottom, and the
upper-most impeller is spaced 1.5 or more impeller diameters from the liquid
surface
Baffling: Stirred tank fermentors generally use baffles because of the need
to disrupt the bulk fluid flow in the tank. Bioreactors do not need this disruption.
In most cases, four flat baffles on 90 centers are used and have a width of .08dt
to .10dt , where dt is the diameter of the tank. For low-viscosity flows baffles are
attached directly to the wall of the tank, but for moderate to high-viscosity flows
baffles are set a small distance away from the wall. While the flat, four-baffle
configuration is most common, other sizes, shapes and number of baffles have
been researched, but only on a limited basis.
Tank Height: The height to diameter ratio of the tank is typically between
2.0 and 3.0; however, taller tanks (up to HL/dt=4.0) have been used to reduce
the power requirement of the impellers. Typical tanks also employ a dish-shaped
bottom to enhance mixing and prevent dead zones
AIRLIFT BIOREACTORS
Most industrial airlift fermenters are of the concentric draft tube type. In
most designs, the coaxial draft tube functions as the aerated section. Air-sparged
liquid rises up the draft tube, is partially degassed and flows down the annulus. In
some designs, the flow pattern is reversed by sparging the air at the base of the
annulus between the draft tube and the outer wall. The choice appears to be a
matter of individual preference, although differences in fermenter foaming and
heat transfer rate have been noted by some investigators.
5
Draft tube diameter: The ratio of the diameter of the draft tube to that of
the fermenter, or the ratio of the cross-sectional area of the riser to that of the
downcomer, are important design characteristics in maximizing liquid circulation
rate and in minimizing mixing time. Diameter ratios in the range of 0.6-0.8 have
often been quoted to give optimal performance.
Draft tube height: Another feature of industrial fermenters of the concentric
draft tube type is their slim design, i.e. very large height to diameter ratios, as large
as 10. Increasing the hydrostatic head by increasing liquid height in the fermenter
increases the partial pressure of oxygen at the base of the aerated section and
provides a larger driving force for oxygen transfer. A large difference in
hydrostatic head between the aerated section and the downcomer section also
promotes liquid circulation. To redisperse gas bubbles in the upflowing fluid,
baffles are sometimes placed in the draft tube
6
In an airlift fermenter, mixing is accomplished without any mechanical

agitation. An airlift fermenter is used for tissue culture, because the tissues are
shear sensitive and normal mixing is not possible. With the airlift, because the shear
levels are significantly lower than in stirred vessels, it is suitable for tissue culture.
The gas is sparged only up to the part of the vessel cross section called the riser.
Gas is held up, fluid density decreases causing liquid in the riser to move upwards
and the bubble-free liquid to circulate through the down-comer. The liquid
circulates in airlift reactors as a result of the density difference between riser and
down-comer.
There are many forms of airlift bioreactor. In the usual form, air is fed into the
bottom of a central draught tube through a sparger ring, so reducing the
apparent density of the liquid in the tube relative to the annular space within the
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bioreactor. The flow passes up through the draught tube to the head space of
the bioreactor, where the excess air and the by-product, CO2, disengage. The
degassed liquid then flows down the annular space outside the draft to the
bottom of the bioreactor. In general, airlift bioreactors have the following
features:
Ideal Bioreactors
In reactor design, the primary aim is to know what should be the type and
size of the reactor, and what methods of operation are best suited for a given job.
The objective behind the reactor design is to design an optimum size of the
reactor for getting maximum yield of the product with highest selectivity. The
conditions in the reactor may vary either with time or position. The conditions
generally include composition and temperature. In addition to these, the
geometry of the reactor will determine the path of the flow of the fluids in the
reactor, which in turn determines the mixing pattern in the reactor.
In this document, the focus is on deciding the best possible conditions for
conducting a bioreaction. Productivity in a reactor of a given volume is of
considerable importance for the commercial value of a bioprocess, sometimes
more important than the yield and the nal titer (concentration) of the desired
product. Thus, we shall discuss the optimal combination of equipment (the
bioreactor) and the environmental conditions, e.g., the dilution rate D, with the
purpose of designing the best possible overall process (highest yield, productivity,
and nal titer), while considering the variable and the xed production costs of
the process.
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This survey of the practical feasibility of bioproduction is to be done by

involving the material of all the preceding chapters together with an engineering
approach to the subject.
Table 1 is an overview of the reactors used in the bioindustry where, in very
general terms, the application areas as well as advantages/disadvantages of the
reactors are stated. By far the most commonly used bioreactor is the stirred tank
reactor (STR). Steady-state operation is the preferred mode of operation of the
STR since the CSTR can be designed to give either maximum productivity or
maximum yield of the desired product, and the operating conditions can be kept
at the set-point for very long times with the use of state-of-the-art process control
equipment which is now obligatory in the bioindustry.
The batch reactor and the fed-batch reactor both operate in non-steady
state, but it is important to realize that the culture is nearly in metabolic steady
state during cultivation in these reactors: The changes in the environment during
the process are often sufciently slow to justify that, in terms of the metabolic state
of the cells, the non-steady-state operating STR is not different from the CSTR.
True dynamic behavior of the culture is only experienced after sudden
changes in the substrate concentration level in the reactor or after sudden
changes in culture parameters, such as the pH. Here, kinetics that are applicable
in the design of processes where the culture is in metabolic steady state cannot
be used as it is impossible to set up a general model that would predict the cell-
level changes of metabolism after a large upset of the steady state. Hence, true
transients are combated with the help of efcient process control equipment,
and the effect of large transients which may have disastrous effects on the
process are only seen after a failure in the process control system (or as a result of
human errors).
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The plug ow reactor (PFR) is rarely used in bioprocessing. If, however, a

valuable gaseous substrate (e.g., technically pure (9597%) O2) is used in a
continuous cultivation, too much of the gaseous substrate is lost when the gas is
simply sparged to the bottom of a STR. The solution is to keep the liquid phase
substrates and products in (almost) completely mixed state, e.g., with a typical
holding time of 45 h, while consuming the gas-phase substrates in plug ow
mode where a satisfactory conversion can be obtained, also with a holding time
of only 2040 s. The equipment used is called a loop-reactor.
STIRRED TANK REACTOR (BATCHQ)

A general mass balance for a STR is,
(1)
On the right-hand side of (1), we nd the rate of transport qt of substrates

(or products) from the gas phase to the liquid medium and the rate of production
of substrates (or products) by the bioreaction. Both are in units of, e.g., kg (m3
reactor)-1h-1. When multiplied by V, we obtain the sum of contributions of reaction
components transported from the gas to the medium and the reactants
produced in the reactor (kg h-1). Also on the right-hand side of (1), we have the
difference between the inlet ow vfcf and efuent ow vece of reaction
components (kg h-1). On the left-hand side of (1) is the accumulation of reactants
in the reactor (kg h-1).
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Fig. 9.1 Schematic representation of the stirred tank reactor
Figure 1 is a schematic representation of the STR showing inputs and

outputs. Both in the CSTR and in the batch reactor, V is constant. In the batch
reactor, both ow terms on the right-hand side of (1) are zero, and in the fed-
batch reactor vece is zero, but V is a function of time t, and most of the variables
in c change with time, although we often desire to use a fed-batch strategy
where s is constant.
In a CSTR with cell retention xe is different from x, the cell concentration in
the reactor, while the other components of ce have the same values of c as in
the reactor.
The control strategy to obtain steady-state conditions in the continuous
ow reactor has usually been to maintain the dilution rate D at a constant value
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by manipulating the ow rate v to keep V or the weight of the medium in the

reactor as constant, i.e., chemostat operation
(2)
CONTINUOUS STIRRED TANK REACTOR

In designing a bioreactor, material balance is used for all the streams
associated with the fermentation vessel. The biomass at inlet, outlet and the
generated biomass must be balanced while the fermentation proceeds. The cell
balance without any cell accumulation is shown in the following equation:
where X is viable cell in the effluent

stream and Xo is viable cell in the feed stream,
F is the volumetric flow rate, V is the reactor
working volume, and rx is the rate of cell
formation per unit volume. The rate equation is
explained in detail by a Monod rate model.
The Monod rate equation is well known in
microbial growth kinetics:
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where m is the specific growth rate, umax is the maximum specific growth
rate, and Ks is the Monod constant.
At steady-state condition for chemostat operation, change of

concentration is independent of time. Material balance for the fermentation
vessel is:
In - Out + Reaction rate = Accumulation
At steady-state condition there is no accumulation, therefore the material

balance is reduced to:
where Cif is the molar concentration in feed stream, Ci is the molar

concentration in the outlet stream and is the rate of feed stream or
consumption rate
The rate of formation of a product is easily evaluated at steady-state

condition for inlet and outlet concentrations, where D is the dilution rate, defined
as , which characterizes, the inverse retention time in the CSTR unit. The
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dilution rate is equal to the number of fermentation vessel volumes that pass
through the vessel per unit time. D is the reciprocal of the mean residence time.
Rate of cell accumulation (dry weight):
In a continuous culture:
Where u is the monod rate of cell growth, and a is the cell death constant
Monod Model for a Chemostat

A Monod rate model is used to demonstrate the rate of biomass
generation. We neglect the cell death rate. Let us denote the ratio of biomass
rate of generation to biomass concentration, rx/X, that is the specific growth rate;
m also denotes the dilution rate; D is defined as number of tank volumes passed
through per unit time, F/V. After substitution of D and m into the following equation
is obtained:
Substituting specific growth rate based on the Monod rate equation, the
rearranging results in:
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The Monod Equation:
For sterile media with suitable nutrients in absence of any organisms,
Biomass generated is considered as
At steady state, substrate utilization is balanced with a rate equation
When the volume of the vessel is divided by the flow rate, retention time
and dilution rate are defined in the following equation:
Combining the last 2 equations:
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YIELD
Let us define yield factor, Y:
CONTINUOUS STIRRED TANK REACTOR WITH RECYCLE

With cell recycling, chemostat efficiency is improved. To maintain a high
cell density the cells in the outlet stream are recycled back to the fermentation
vessel. The figure below represents a chemostat unit with a cell harvesting system.
The separation unit is used for harvesting the cells and recycling then to the
culture vessel to increase the cell concentration. The material balance in a
constant volume chemostat is derived based on cell balance as shown in the
following equations. Material balance in a chemostat with recycle,
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For steady-state
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MULTI-STAGE CSTR
Multi-stages of continuous culture are designed to use the outlet of the first
vessel as the inoculum for the next stage. If intermediate metabolites are used as
feed for another microorganism, sequential continuous culture is useful. The
dilution rate for each vessel may be different to the other vessel. It is also possible
to supply different nutrients for each stage of fermentation vessel. It is common to
operate earlier stages as aerobic and subsequent stages in an anaerobic
condition. In addition, if unused substrate leaves the product stream, it can be
used in the next stage even at low substrate concentration. The kinetic
representation may show a slower rate and even drop to zero-order. Figure 5.11
shows two stages of a chemostat in operation.
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FED-BATCH REACTORS
Culture with continuous nutrient supply can be operated in two modes: (i)
variable volume; (ii) fixed volume. For variable volume, feed rate Fin is not the
same as outflow when Fout=0
and dilution rate,
Material balance for substrate:
For steady-state
Substituting u:
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PLUG FLOW REACTOR

When fluid moves through a large pipe or channel with sufficiently large
Reynolds number (e.g., > 2100 in a pipe), it approximates plug flow, which means
that there is no variation of axial velocity over the cross section. If it is assumed
that plug flow approximately describes fluid movement through the reactor, the
mass balance on the plug-flow tubular reactor can be (PFTR) easily formulated
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CSTR WITH ENZYME-CATALYZED REACTION

The Michaelis-Menten equation states that
where S is the substrate and Vmax = kcat [Et]

KM is equal to the substrate concentration at which the rate of reaction is equal
to one-half the maximum rate. To linearize the Michaelis-Menten equation (called
the Lineweaver-Burk plot)
For a batch enzymatic reaction,
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Sterilization
Most industrial fermentations are carried out as
pure cultures in which only selected strains are
allowed to grow. If foreign microorganisms exist in the
medium or any parts of the equipment, the
production organisms have to compete with the
contaminants for the limited nutrients. The foreign
microorganisms can produce harmful products
which can limit the growth of the production organisms. Therefore, before starting
fermentation, the medium and all fermentation equipment must be free from any
living organisms; in other words, they have to be completely sterilized.
Furthermore, the aseptic condition has to be maintained.
BATCH STERILIZATION
Batch sterilization is the reduction of contaminant organisms through the
heating of a vessel. The entire volume of media is sterilized at once through the
use of thermal or radiation techniques. When running a thermal batch sterilization,
a system goes through 3 steps: heating, holding, and cooling. Heating requires
the addition of energy throughout the entire medium volume. This can be done
by adding heat through a jacket on the vessel. The temperature is increased until
it reaches the sterilization temperature where it is held for a set period of time.
During this phase, most of the unwanted microorganisms are destroyed. Finally,
the system is cooled to bring the sterile media back to the desired temperature.
For radiation sterilization, the process is similar to above, although it uses radiation
22
intensity instead of heat. Del factor is a measure of fractional reduction of viable

organism count produced by a certain heat and time regime
In order to sterilize a batch, calculate the total area underneath the curve.
Therefore, model death using first order kinetics and integrate as seen above. This
will yield a temperature and the corresponding duration of time needed to
sterilize the media.
Advantages:
Most widely used technique
Simple operation
No additional materials are added to the media itself
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Disadvantages:
More expensive heat requirements than continuous sterilization
Best results occur in well-mixed closed vessels
CONTINUOUS STERILIZATION
Continuous sterilization is the rapid transfer of heat to medium through
steam condensate without the use of a heat exchanger. Once the media is in a
holding loop, steam is injected to the system via a nozzle. The medium stays in this
loop for a predetermined holding time until the entire medium is sterile. This is more
efficient than batch sterilization because instead of expending energy to heat,
hold, and cool the entire system, small portions of the inlet streams are heated at
a time. By looping sterile media tubes (which are at higher temperatures) past
inlet tubes, the difference in temperature is used to help heat the unsterile
medium. So instead of having a cold-water stream cool the sterile media, the
lower temperature unsterile media stream absorbs heat from the warm stream,
cooling the sterile media. Finally, the sterile media is flash cooled through an
expansion valve to adjust the temperature to meet process parameters.
24
Advantages:
Uniform steam requirements throughout the duration of the sterilization
Simplified process control
Shorter sterilization time means less thermal degradation of medium
Disadvantages:
High demand for steam in a shorter period of time than batch
Concentration of media becomes dilute due to steam condensation
Since steam is actually dispersed in media, steam must be clean to
avoid contamination
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PAMANTASAN NG LUNGSOD NG MAYNILA

University of the City of Manila

DESIGN AND ANALYSIS

Bioreactor Analysis and Design

General Design Guide

Fluidization and Dust & Mist Collection

Height of vessel to diameter:

Fluidization and Dust & Mist Collection

Diameter of vessel to baffle:

Fluidization and Dust & Mist Collection

Fluidization and Dust & Mist Collection

Fluidization and Dust & Mist Collection

In an airlift fermenter, mixing is accomplished without any mechanical

This survey of the practical feasibility of bioproduction is to be done by

The plug ow reactor (PFR) is rarely used in bioprocessing. If, however, a

STIRRED TANK REACTOR (BATCHQ)

On the right-hand side of (1), we nd the rate of transport qt of substrates

Fluidization and Dust & Mist Collection

Fig. 9.1 Schematic representation of the stirred tank reactor

Figure 1 is a schematic representation of the STR showing inputs and

Fluidization and Dust & Mist Collection

by manipulating the ow rate v to keep V or the weight of the medium in the

CONTINUOUS STIRRED TANK REACTOR

where X is viable cell in the effluent

Fluidization and Dust & Mist Collection

At steady-state condition for chemostat operation, change of

At steady-state condition there is no accumulation, therefore the material

where Cif is the molar concentration in feed stream, Ci is the molar

The rate of formation of a product is easily evaluated at steady-state

Fluidization and Dust & Mist Collection

Rate of cell accumulation (dry weight):

Monod Model for a Chemostat

Fluidization and Dust & Mist Collection

The Monod Equation:

For sterile media with suitable nutrients in absence of any organisms,

Biomass generated is considered as

At steady state, substrate utilization is balanced with a rate equation

Combining the last 2 equations:

Fluidization and Dust & Mist Collection

CONTINUOUS STIRRED TANK REACTOR WITH RECYCLE

Fluidization and Dust & Mist Collection

Fluidization and Dust & Mist Collection

Fluidization and Dust & Mist Collection

and dilution rate,

Material balance for substrate:

Fluidization and Dust & Mist Collection

PLUG FLOW REACTOR

Fluidization and Dust & Mist Collection

CSTR WITH ENZYME-CATALYZED REACTION

where S is the substrate and Vmax = kcat [Et]

For a batch enzymatic reaction,

Fluidization and Dust & Mist Collection

intensity instead of heat. Del factor is a measure of fractional reduction of viable

Fluidization and Dust & Mist Collection

Fluidization and Dust & Mist Collection

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