CP504 – ppt_Set 10
Sterilization (continued)
- Learn about thermal sterilization of liquid medium
- Learn about air sterilization (only the guidelines)
- Learn to do design calculations
R. Shanthini
Feb 01, 2013
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Continuous Sterilization:
- Simplifies production planning
- Therefore gives maximum plant utilization and minimum
delays
- Provides reproducible conditions
- Can be operated at high temperature (1400C)
- Therefore sterilization time can be reduced (2 to 3 min)
- Requires less steam and less cooling water
- Suitable when the capacity of operation is high
- High initial capital investment (use of aseptic transfer system
for the sterile broth to be transported to a sterile vessel)
R. Shanthini
Feb 01, 2013
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Continuous Sterilization:
It is the only option if the medium is to be exposed to high
temperatures for a short time (HTST process) to avoid
denaturation of proteins or to avoid destruction of some of
the enzymes, etc., since it is not possible in commercial
scale operations in batch sterilization to quickly heat large
volumes of the medium in short time and cool it also in short
times.
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Feb 01, 2013
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Continuous Sterilization:
Continuous Injection Type
Steam
Raw
medium
Expansion
valve
Holding section
(where most of the
sterilization takes place)
vacuum
Flash
cooler
- Direct steam injection for heating
(relatively a rapid process)
- Flash cooling (rapid process)
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Feb 01, 2013
Sterile
medium
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Continuous Sterilization:
Continuous Injection Type
- Capital investment is low
- Easy to clean and maintain the system
- Heating and cooling periods are shorter
- Steam efficiency is very high as live steam is directly injected
into the medium
- Direct contact of the steam with the medium makes it
necessary that the steam should be clean and free of any anticorrosive agents
- Foaming may occur during both heating and cooling
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Feb 01, 2013
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Continuous Sterilization:
Sterile
medium
Continuous Heat Exchanger Type
Steam
Holding section
Cooling water
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Feb 01, 2013
Raw medium
Indirect steam
heating in
plate-and-frame (or
shell-and-tube)
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heat exchanger
Continuous Sterilization:
Continuous Heat Exchanger Type
plate-and-frame heat exchanger has larger heat transfer area
than shell-and-tube heat exchanger, and therefore more
effective
plate-and-frame heat exchanger is favourable with high
viscous system
plate-and-frame heat exchanger is limited to lower pressures
(less than 20 atm) due to its weak structural strength
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Feb 01, 2013
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Continuous Sterilization:
Most of the sterilization in the continuous sterilization process
may occur in the holding section.
Therefore
hold =
no
ln
nt
= kd λhold = kd0 exp(-Ed/RT) λhold
Since the holding section is a long pipe,
λhold
=
L
uav
length of the pipe
average fluid velocity
in the pipe
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Feb 01, 2013
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Continuous Sterilization:
The ratio of average velocity to maximum velocity
uav
umax
=
0.5 for laminar flow of Newtonian fluids
through a smooth round pipe
=
0.75 for turbulent flow
=
0.87 for turbulent flow with the Reynolds
Number of 1000,000
Therefore, using the average velocity to calculate
the length of the pipe required for sterilization may
leave some portion of the medium understerilized,
which may cause contamination problem
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Feb 01, 2013
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Example 10.4: Estimating the time required for continuous sterilization
A continuous sterilizer with a steam injector and a flash cooler
will be employed to sterilize medium continuously with the flow
rate of 2 m3/h. The time for heating and cooling is negligible with
this type of sterilizer. The typical bacterial count of the medium is
about 5 x 1012 per m3, which needs to be reduced to such an
extent that only one organism can survive during two months of
continuous operation. The sterilizer will be constructed with the
pipe with an inner diameter of 0.102 m. Steam at 600 kPa
(gauge pressure) is available to bring the sterilizer to an
operating temperature of 125oC. For the heat resistant bacterial
spores: kdo = 5.7 x 1039 per h; Ed = 2.834 x 105 kJ / kmol. For the
medium: c = 4.187 kJ/kg.K; ρ = 1000 kg/m3; μ = 4 kg/m.h.
a) What length should the pipe be in the sterilizer if you assume
ideal plug flow?
b) What length should the pipe be in the sterilizer if the effect of
axial dispersion is considered? Assume an axial dispersion
coefficient
of 20 m2/h.
R.
Shanthini
10
Feb 01, 2013
Solution to Example 10.4:
Problem statement: The typical bacterial count of the medium
is about 5 x 1012 per m3, which needs to be reduced to such
an extent that only one organism can survive during two
months of continuous operation.
n0 = (5 x 1012 per m3) x (2 m3/h) x (24 h/day) x (60 days)
= 14400 x 1012
nt = 1

=
t
n0
ln
nt
=
144x1014
= 37.2 =
ln
1

kd dt
0
The above integral should give 37.2.
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Feb 01, 2013
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Solution to Example 10.4:
a) Since the temperature at the holding section in constant,
hold
= kd λhold = 37.2
For the given data,
kd = kd0 exp(-Ed/RT)
= (5.7 x 1039 per h) exp[- 2.834x105 / 8.314x(273.15+125)]
= 375.3 per h
Therefore, λhold = 37.2 / kd = 37.2 / 375.3 per h = 0.099 h
Length of the pipe, L = velocity through the pipe x λhold
= velocity through the pipe x 0.099 h
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Feb 01, 2013
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Solution to Example 10.4:
Since plug flow is assumed,
velocity through the pipe =
2 m3/h
π (0.102/2)2 m2
= 245 m/h
Therefore, L = (245 m/h) x (0.099 h) = 24.26 m
What happens if the flow is not plug flow?
R. Shanthini
Feb 01, 2013
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Solution to Example 10.4:
b) If axial dispersion is considered, then we use the following
table:
uav L
Peclet number =
D
nt
n0
R. Shanthini
Feb 01, 2013
Axial dispersion
coefficient
kd L
uav
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Solution to Example 10.4:
=
(245 m/h) x ( L m)
(20 m2/h)
X-coordinate =
kd L
uav
=
(375.3 per h) x ( L m)
(245 m/h)
Y-coordinate =
nt
n0
is obtained from the table
uav L
Peclet number =
D
R. Shanthini
Feb 01, 2013
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Solution to Example 10.4:
If L = 25 m is tried, we get
uav L
Peclet number =
D
=
(245 m/h) x ( 25 m)
(20 m2/h)
X-coordinate =
kd L
uav
=
Y-coordinate =
nt
n0
=
= 306
(375.3 per h) x ( 25 m)
= 38.3
(245 m/h)
2 x 10-15
Data provided: n0 = 14400 x 1012; nt = 1
Therefore, required nt / n0 = 6.9 x 10-17
R. Shanthini
Feb 01, 2013
Calculated ratio > Required ratio
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Solution to Example 10.4:
If L = 27.5 m is tried, we get
=
(245 m/h) x ( 27.5 m) = 337
(20 m2/h)
X-coordinate =
kd L
uav
=
(375.3 per h) x ( 27.5 m)
= 42.1
(245 m/h)
Y-coordinate =
nt
n0
≈ 6.9 x 10-17
uav L
Peclet number =
D
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Feb 01, 2013
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Filtration:
Sterilize solutions that may be damaged or denatured by
high temperatures or chemical agents.
The pore size for filtering bacteria, yeasts, and fungi is in
the range of 0.22-0.45 μm
The pore size for filtering viruses and some large proteins
is in the range of 0.01 μm
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Feb 01, 2013
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Methods for gas (air) sterilization:
- Aerobic fermentation require huge volumes of air (a
50,000 L fermenter requires 7x106 to 7x107 L per day
of air) which must be sterilized.
- Adiabatic compression of process air can increase air
temperature (150oC to 220oC). A temperature typically
of 220oC for 30 s is required to kill spores.
- An air-filtration step is almost always used to ensure
sterility of process air.
- Depth filters use glass wool, and rely on inertial
impaction, interception, diffusion and electrostatic
attraction (explained later).
- Surface filters using membrane cartridges use the
sieving effect (explained later).
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Feb 01, 2013
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Filtration mechanisms operating in depth filters
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Feb 01, 2013
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http://blogs.cdc.gov/niosh-science-blog/2009/10/n95/
Filtration mechanisms operating in depth filters
Inertial impaction (or impingement) occurs when a particle
travelling in the air stream and passing around a fibre,
deviates from the air stream (due to particle inertia) and
collides with the fibre.
Impaction is the dominant collection mechanism for
particles larger than 0.2 μm.
It is important in removing bacteria.
R. Shanthini
Feb 01, 2013
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Filtration mechanisms operating in depth filters
Interception occurs when a large particle, because of its
size, collides with a fibre in the filter that the air stream is
passing through.
Interception is the dominant collection mechanism for
particles greater than 0.2 μm.
It is important in removing bacteria.
R. Shanthini
Feb 01, 2013
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Filtration mechanisms operating in depth filters
Diffusion occurs when the random (Brownian) motion of a
particle causes that particle to contact a fibre.
Diffusion is dominant for particles less than 0.2 μm.
It may be important for virus removal, but bacteria are
sufficiently large that diffusion is relatively unimportant.
R. Shanthini
Feb 01, 2013
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Filtration mechanisms operating in depth filters
Electrostatic attraction plays a very minor role in mechanical
filtration. After fibre contact is made, smaller particles are
retained on the fibres by a weak electrostatic force.
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Feb 01, 2013
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Filtration mechanisms operating in surface filters
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Feb 01, 2013
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http://www.renewableenergy.nl/index.php?pageID=3220&n=545&itemID=351064
Filtration mechanisms operating in surface filters
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Feb 01, 2013
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http://www2.donaldson.com/torit/en-us/pages/technicalinformation/mistcollection_fundandapp.aspx
A typical bioreactor used for microbial fermentations:
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Feb 01, 2013
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Stirred tank bioreactor – Oxygen delivery system:
The oxygen delivery system consists of
 a compressor
 inlet air sterilization system
 an air sparger
 exit air sterilization system
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Feb 01, 2013
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Stirred tank bioreactor – Oxygen delivery system:
• A compressor forces the air into the reactor. The
compressor will need to generate sufficient pressure to
force the air through the filter, sparger holes and into the
liquid.
• Air compressors used for large scale bioreactors
typically produce air at 250 kPa. The air should be dry
and oil free so as to not block the inlet air filter or
contaminate the medium.
• Note that it is very important that an "instrument air"
compressor is not used. Instrument air is typically
generated at higher pressures but is aspirated with oil.
Instrument air compressors are used for pneumatic
control.
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Feb 01, 2013
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Stirred tank bioreactor – Air sterilization:
• Sterilization of the inlet air is undertaken to prevent
contaminating organisms from entering the reactor.
• The exit air on the other hand is sterilized not only to
keep contaminants from entering but also to prevent
organisms in the reactor from contaminating the air.
• A common method of sterilising the inlet and exit air is
filtration.
• For small reactors (with volumes less than 5 litres), disk
shaped hydrophobic Teflon membranes housed in a
polypropylene housing are used. Teflon is tough,
reusable and does not readily block.
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Feb 01, 2013
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Stirred tank bioreactor – Air sterilization:
• For larger laboratory scale fermenters (up to 1000 litres),
pleated membrane filters housed in polypropylene
cartridges are used.
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Feb 01, 2013
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Stirred tank bioreactor – Air sterilization:
• By pleating the membrane, it is possible to create a
compact filter with a very large surface area for air filtration.
Increasing the filtration area decreases the pressure
required to pass a given volume of air through the filter.
• Sterilization of the inlet and exit air in large bioreactors (>
10,000 litres) can present a major design problem. Large
scale membrane filtration is a very expensive process. The
filters are expensive as they are difficult to make and the
energy required to pass air through a filter can be quite
considerable.
R. Shanthini
Feb 01, 2013
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Stirred tank bioreactor – Air sterilization:
• Heat sterilization is alternative option.
• Steam can be used to sterilize the air.
• With older style compressors, it was possible to use the
heat generated by the air compression process to sterilize
the air.
• However, compressors are now multi-stage devices which
are cooled at each stage and disinfecting temperatures are
never reached.
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Feb 01, 2013
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Air sterilization – positive pressure:
• During sterilisation, the concept of "maintaining positive
pressure" will often be used.
• Maintaining positive pressure means that during
sterilisation, cooling and filling and, if appropriate, the
fermentation process, air must be pumped into the reactor.
• In this way the reactor is always pressurised and thus
aerial contaminants will not be "sucked" into the reactor.
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Feb 01, 2013
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Air sterilization – positive pressure:
Without aeration,
a vacuum forms
as the reactor
cools.
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Feb 01, 2013
With aeration, positive
pressure is always
maintained and
contaminants are pushed
away from the reactor
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Stirred tank bioreactor - Cleaning and sterilization facilities:
• Small scale reactors are taken apart and then cleaned before
being re-assembled, filled and then sterilized in an
autoclave.
• However, reactors with volumes greater than 5 litres cannot
be placed in an autoclave and sterilized. These reactors
must be cleaned and sterilized "in place". This process is
referred to "Clean in Place” (CIP).
• CIP involves the complete cleaning of not only the fermenter
but also all lines linked to the internal components of the
reactor. Steam, cleaning and sterilizing chemicals, spray
balls and high pressure pumps are used in these processes.
The process is usually automated to minimize the possibility
of human error.
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Feb 01, 2013
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Concerns in air sterilization:
Pressure drop is critical in a filter.
Energy input for compressed air for a commercial-scale
process is significant.
Air treatment can account for 25% of total production
costs.
Design engineer has to balance the assurance sterility
against the pressure drop.
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Feb 01, 2013
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180M3 Fermenter Plant
Air Compressor
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Feb 01, 2013
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Air Filters in Fermentation Plant
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Feb 01, 2013
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sterilization note from Lee was handed over.
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