Sterilization BioChem
Sterilization BioChem
Sterilization BioChem
Introduction
Before starting fermentation, the medium and
all fermentation equipment have to be free
from any living organisms, in other words they
have to be completely sterilized. Furthermore,
the aseptic condition has to be maintained.
Sterilization Methods
Sterilization of fermentation media or equipment
can be accomplished by destroying all living
organisms by:
(a) means of heat (moist or dry)
(b) chemical agents
(c) radiation (ultraviolet or X-rays)
(d) mechanical means (sonic or ultrasonic
vibrations)
(e) Filtration or high-speed centrifugation
n
ln
k d dt
n0
n n0 exp k d dt
Linear Form
Ed
k d k d 0 exp
RT
Ed 1
ln k d ln k d 0
R T
Design Criterion
The design criterion, , for sterilization
(Deindoerfer and Humphrey, 1959) is given by
t
n0
Ed
ln k d dt k d 0 exp
dt
n
RT
0
0
Batch Sterilization
Sterilization of the medium in a fermenter can
be carried out in batch mode by direct steam
sparging, by electrical heaters, or by constant
pressure condensing steam. The sterilization
cycles are composed of heating, holding, and
cooling. Therefore, the total Del factor
required should be equal to the sum of the
Del factor for heating, holding, and cooling as
Direct steam
sparging
R. Shanthini
18 Nov 2011
Electrical
heating
Steam
heating
9
Batch Sterilization
The design procedure for the estimation of the
holding time is as follows:
1. Calculate the total sterilization criterion, total.
2. Measure the temperature versus time profile
during the heating, holding, and cooling cycles
of sterilization. The suggested theoretical
equations for different heating and cooling
processes are as follows (Deindoerfer and
Humphrey, 1959):
Batch Sterilization
a. For batch heating by direct steam sparging
into the medium, the hyperbolic form is
used:
Hms t
T T0
cM ms t
b. For batch heating with a constant rate of heat
flow, the linear form is used:
T T0
qTt
cM
Batch Sterilization
c. For batch heating with isothermal heat
source, the exponential form is used:
UAt
T TH T0 TH exp
cM
UA mc t
T TC 0 T0 TC 0 exp1 exp
m
c
M
Batch Sterilization
3. Plot the values of kd as a function of time.
4. Integrate the areas under the kd-versus-time
curve for the heating and the cooling periods
to estimate heat and cool, respectively. The
holding time can be calculate from
hold total heat cool
thold
kd
kd
Example 10.1:
R. Shanthini
18 Nov 2011
kd = 0.7413
Sf = n/n0 = 1.32531013
Spore
density
(m-3)
10
15
20
30
8.5x1010
4.23x109
6.2x107
1.8x106
4.5x104
32.5
Example 10.2:
Ed = 294,745.9627 J/mol
kd0 = 1.62431038 min1
kd @ 130C = 1.0475 min1
R. Shanthini
18 Nov 2011
115
120
125
0.035
0.112
0.347
Supplementary Problem:
Reference: Biochemical Engineering / Shigeo Katoh and Fumitake Yoshida
Example 8.1
A fermenter containing 40 m3 of medium (25C)
is going to be sterilized by the direct injection
of saturated steam. The typical bacterial count
of the medium is about 51012 m3, which
needs to be reduced to such an extent that
the chance for a contaminant surviving the
sterilization is 1 in 1,000. The steam (345 kPa,
absolute pressure) will be injected with a flow
rate of 5,000 kg/h, which will be stopped
Example 8.1
when the medium temperature reaches 122C.
During the holding time, the heat loss through
the vessel is assumed to be negligible. After
proper holding time, the fermenter will be
cooled by passing 100 m3/h of 20C water
through the cooling oil in the fermenter until
the medium reaches 30C. The coil has a heattransfer area of 40 m2 and for this operation
the average
Example 8.1
overall heat-transfer coefficient (U) for cooling is
2,500 kJ/hm2K. The heat-resistant bacterial
spores in the medium can be characterized by
an Arrhenius coefficient (kd0) of 5.71039 h1
and an activation energy (Ed) of 2.834105
kJ/kmol (Deindoerfer and Humphrey, 1959).
The heat capacity and density of the medium
are 4.187 kJ/kgK and 1,000 kg/m3,
respectively. Estimate the required holding
time.
Supplementary Problem:
Reference: Biochemical Engineering / Shigeo Katoh and Fumitake Yoshida
Continuous Sterilization
Advantages of continuous sterilization are:
1. It simplifies production planning, thus allowing
maximum plant utilization and minimum delays.
2. It provides reproducible conditions.
3. It can be operated at a high temperature (140C
instead of 121C in batch sterilization);
therefore, the sterilization time can be
shortened (holding time of 1 to 2 minutes).
Continuous Sterilization
4. It requires less steam by recovering heat from
the sterilized medium. As a result, it also
requires less cooling water.
5. It is easier to automate the process; thus, it is
less labor intensive.
A continuous sterilizer consists of three main
sections: heating, holding, and cooling. The
following figure shows typical continuous
sterilization units.
Continuous Sterilization
Continuous Sterilization
Continuous Sterilization
HEATING SECTION
Two types:
a. Direct steam injection
b. Indirect heating in shell-and-tube or plateand-frame heat exchanger
Continuous Sterilization
The temperature change with respect to
residence time ( ) as the medium passes
through an isothermal heat source can be
approximated as (Deindoerfer and
Humphrey, 1959b)
TC 2
UA heat
TH TH TC1 exp
cW
Continuous Sterilization
For heating using a countercurrent heat source
of equal flow rate and heat capacity,
TC 2
TUA heat
TC1
cW
Continuous Sterilization
HOLDING SECTION
The holding section is maintained in adiabatic
conditions. If the heat loss in the section is
negligible, the temperature can be assumed
constant. The average residence time in the
holding section is
L
hold
u
Continuous Sterilization
The Del factor can be estimated as
n0
Ed
hold ln kd hold k d 0 exp
hold
n
RT
Continuous Sterilization
Dispersion Model
Given
Continuous Sterilization
Basic material balance for the microorganisms
suspended in medium is
In Out Killed by Sterilization = Accumulation
The input and output of the MOs into or out of
the element have both bulk flow and an axial
diffusion condition. The number of
microorganisms entering minus those leaving
by bulk flow is
d uC
uCn S uCn S
dx
dxS
Continuous Sterilization
The x-directional flux of microorganisms
suspended in a medium due to the axial
mixing can be represented as
dCn
J n D
dx
Continuous Sterilization
The number of MOs entering and leaving by
axial dispersion is
dCn
dCn d
dCn
DS
DS
DS
dx
dx
dx dx
dx
dx dx
dx
Continuous Sterilization
For the constant D and u can be modified into
a dimensionless form,
d 2Cn'
dx'2
Pe
dCn'
dx'
Pe
kd L
u
Cn' 0
where:
Cn'
Cn
Cn 0
x
x
L
'
uL
Pe
D
Continuous Sterilization
The boundary conditions for the differential
equation given is
dCn'
dx
'
at x = 0
at x = 1
Pe 1 Cn' 0
dCn'
dx
'
Continuous Sterilization
The solution is
Cn' x '1
4 exp0.5Pe
where
4k d L / u
1
Pe
Continuous Sterilization
Continuous Sterilization
Continuous Sterilization
COOLING SECTION
The temperature versus residence time
relationship for cooling using an isothermal
heat sink is
TH 2
UA cool
TC TC TH 1 exp
cW
TUA cool
TH 1
cW
Example 8.2
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 51012 m3, which needs to be reduced
to such an extent that only one
microorganism can survive during two
months of continuous operation. The heat-
Example 8.2
resistant bacterial spores in the medium can be
characterized by an Arrhenius coefficient (kd0) of
5.71039 h1 and an activation energy (Ed) of
2.834105 kJ/mol (Deindoerfer and Humphrey,
1959). The sterilizer will be constructed with the
pipe with an inner diameter of 0.102 m. Steam
at 600 kPa (gage pressure) is available to bring
the sterilizer to an operating temperature of
125C. The physical properties of this medium at
125C are c = 4.187 kJ/kgK, = 1000 kg/m3 and
= 4 kg/mh.
Example 8.2
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?
Example 8.2
kd0 = 5.71039 h1
Ed = 2.834105 kJ/kmol
Given
Qm = 2 m3/h
n0 = 51012 m3
c = 4.187 kJ/kgK
= 1000 kg/m3
= 4 kg/mh
dt = 0.102 m
Example 8.2
Required
a. L if ideal plug flow
b. L if effect of axial dispersion is considered
Example 8.2
Solution
a. The design criterion is given by
5 1012 m3 2 m3 /h 24 h/d60 d
n0
ln ln
37.2060
n
1
k d hold
Example 8.2
Solution
kd is given by
Ed
k d k d0 exp
RT
5.7 1039 h1
2.834
10
J/mol
exp
8.314J/mol K 125 273.15K
375.2590h
Example 8.2
The residence time is
hold
37.2060
0.0991h
1
k d 375.2590h
244.7596m/h
2
2
A
/4 0.102 m
Example 8.2
The length of the sterilizer is
L uhold 244.7596m/h0.0991 h 24.0599 m
dt u 0.102m244.7596m/h 1000kg/m3
Re
4 kg/m h
6,241.3698 6.24 10
Example 8.2
From Figure 8.4
D
0.8
udt
for Re = 6.24103
Therefore,
D 0.8udt 20 m /h
2
Example 8.2
Assume L = 25 m, the length for ideal plug flow,
then
uL 24525
Pe
306
D
20
and
378.625
38.7
245
u
k dL
Example 8.2
From Figure 8.5, for Pe = 306 and k dL/u 38.7,
Cn
2 1015
Cn0
Air Sterilization
In aerobic fermentations, air needs to be
supplied continuously and it should be noted
that not only the medium should be free of
contaminants but the air to be supplied as
well. Typical aeration rates for aerobic
fermentation are 0.5 1.0 vvm (air volume
per liquid volume per minute). All of the
sterilization techniques discussed for medium
can also be employed for air. However,
Air Sterilization
sterilization of air by means of heat is
economically impractical and is also
ineffective due to the low heat-transfer
efficiency of air compared with those of
liquids. The most effective technique for air
sterilization is filtration using fibrous or
membrane filters.
Examples: (a) cotton plug; (b) glass fibers
Air Sterilization
With fibrous filters, airborne particles are
collected by the mechanisms of impaction,
interception, and diffusion.
Impaction
When an air stream containing particles flows
around a cylindrical collector, the particle will
follow the streamlines until they diverge
around the collector. The particles because of
their mass will have sufficient momentum to
continue to move toward the cylinder and
break through the streamlines, as shown in
the next slide.
Impaction
Impaction
The collection efficiency by this inertial
impaction mechanism is the function of the
Stokes and Reynolds number as:
imp
C f p d 2p vo D v
f St, Re c f
, c o
18Dc
Impaction
The value of Cf can be estimated from the
empirical correlation developed by Davies
(Strauss, 1975).
d p
2
C f 1
1.257 0.400exp 1.10
d p
2
0.499 8RT
Impaction
The efficiency imp is defined as the fraction of
particles approaching the collector which
impact. Various correlations are available in
the literature. An empirical correlation for the
efficiency developed by Thom is (Strauss,
1975):
imp
St 3
St 0.77St 0.22
3
for Rec = 10
Impaction
Another correlation proposed by Friedlander
(1967) is
imp 0.075St
1.2
Interception
The inertial impaction model assumed particles
had mass, and hence inertia, but no size. An
interception mechanism is considered where
the particle has size, but no mass, and so they
can follow the streamlines of the air around
the collector. If a streamline which they are
following passes close enough to the surface
of the fiber, the particles will contact the fiber
and be removed.
Interception
Interception
The interception efficiency depends on the ratio
of the particle diameter to the cylidndrical
collector diameter ( = dp/Dc):
int
2.002 ln Re c
2
1 ln1 21
Interception
The collection efficiency by interception
increases with the increase of particle size.
Diffusion
Particles smaller than about 1 micron in
diameter exhibit a Brownian motion which is
sufficiently intense to produce diffusion. If a
streamline containing these particles is
sufficiently close to the collector, the particles
may hit the collector and be removed.
Contrary to the previous two mechanisms, the
collection efficiency by diffusion increases
with decreasing particle size or air velocity.
Diffusion
The typical size of particles collected by this
mechanismis less than about 0.5 micron. The
efficiency collection by diffusion can be
estimated by an equation analogous to
Langmuirs equation as (Strauss, 1975):
dif
2.002 ln Re c
Z 2 Z
1 Z ln1 Z 21 Z
Diffusion
where Z is the diffusion parameter defined as
1/ 3
DBr
Z 2.242.002 ln Re c
vD
c
Diffusion
where Pe is Pclet number, an important
dimensionless parameter in the theory of
convective diffusion. It is defined as
vo Dc
Pe
Re Sc
DBr
Sc
DBr
Diffusion
The diffusivity due to Brownian movement for
submicron size particles can be estimated
from
DBr
C f kT
3d p
Combined Mechanisms
The total collection efficiency of a fibrous filter is
obtained from the combined effect of the
preceding three mechanisms. One
straightforward way to combine the collection
efficiencies of the different mechanisms is to
add them together, but this implies that a
particle can be collected more than once,
which does not make sense. A better
approach is to use the following correlation:
Combined Mechanisms
which allows only the particles not collected by
one mechanism to be collected by the others.
Pasceri and Friedlander (1960) correlated the
combined collection efficiency as
c
6
Sc 2 / 3Re c1 / 2
3 2 Re c1 / 2
Combined Mechanisms
With an increase of the superficial velocity (vo),
imp and int increase whereas dif decreases.
Therefore, the combined collection efficiency
normally decreases to reach a minimum point
and then increases with increasing superficial
air velocity.
vo
d Cn
v
v
v
1
Cn o A1 Cn o
dh A1 o Cn Adh c Dc L
1
dh
1
1
dh
Cn Dc 1
upon integration yields
Cn
4B
ln
c
Cn 0
Dc 1
c
Dc 1
f 1 4.5
Example 8.3
A filter bed of glass fibers (Dc = 15 m, the bed
depth B = 10 cm, and packing density =
0.03) is being used to sterilize air (20C, 1 atm)
with an undisturbed upstream velocity, vo, of
10 cm/s. the air stream contains 5,000
bacteria per cubic meter (dp = 1 m and p = 1
g/cm3).
Example 8.3
a. Estimate the single fiber collection efficiency by
inertial impaction, by interception, and by
diffusion.
b. Estimate the single fiber collection efficiency
based on combined mechanisms by using Eq.
(8.42) and Eq. (8.43) and compare the results.
c. Estimate the collection efficiency () of the
filter bed.
d. Show how superficial velocity vo affects the
various single fiber collection efficiencies.
Example 8.3
Given
Air
@ 20C,
1 atm
vo = 10 cm/s
dp = 1 m
p = 1 g/cm3
Dc = 15 m
B = 10 cm
= 0.03
Sterilized air
Example 8.3
Required
a. imp, int and dif
b. c by Eq. 8.42 and Eq. 8.43 and compare
results
c.
d. show how vo affects various single fiber
collection efficiencies
Example 8.3
10.3093 cm/s
1 1 0.03
Example 8.3
PM
1 atm29 g/mol
1L
3
L
atm
RT
1000 cm
0.08205
20 273.15K
mol K
1.2057 10 3 g/cm3
air 1.810 4 g/cm s
0.1036
1.80 10
Example 8.3
The mean free path and the Cunningham correction factor Cf can be
estimated from Eqs. 8.32 and 8.31
Mw
1.80 10 4
3
0.499 8RT 0.499 1.20 10
29
6.50 10 6 cm
dp
2
Cf 1
1.257 0.400exp 1.10
dp
2
2 6.50 10 6
1
10
Cf 1
1.257 0.400exp 1.10
4
-6
1 10
2
6.50
10
1.16
Example 8.3
The mean free path and the Cunningham correction factor Cf can be
estimated from Eqs. 8.32 and 8.31
The Stokes number is
St
C f p d 2p v
18Dc
1.161 1 10 4 10.3
18 1.80 10
1.510
3
0.0247
Example 8.3
The single fiber collection efficiency by interception is from Eq. 8.35 for
= dp/Dc = 0.0667 and Rec = 0.103
int
int
2.002 lnRe c
2
1 ln1 21
1
0.06672 0.0667
0.0667
ln
1
0.0667
2.002 ln0.103
21 0.0667
9.96 10 4
Example 8.3
The diffusivity due to Brownian motion can be estimated from Eq.
8.41,
DBr
C f kT
3d p
3 1.80 10 4 1 10 4
2.77 10 7 cm2 /s
Dc v 1.5 10 3 10.3
4
Pe
5.57
10
DBr
2.77 10 7
The single fiber efficiency by diffusion can be estimated from Eq. 8.38
2/3
dif 1.3Pe
4 2/3
0.70.06672 4.00 10 3
Example 8.3
b. The combined collection efficiency can be estimated from Eq. 8.42
as: c = 1 (1 imp)(1 int)(1 dif)
= 1 (1 8.82104)(1 9.96104)(1 4.00103)
= 0.0059
Instead, if we use Eq. 8.43
1.80 10 4
5
Sc
5.41
10
DBr 1.20 10 3 2.77 10 7
Therefore,
3 2Re1/2
c
Sc 2/3Re1/2
c
5.4110
5 2/3
0.1031/2
Example 8.3
In this example, diffusion is predominant over impaction and
interception. If we use Eq. 8.33 and Eq. 8.36 for the estimation of
imp and dif, respectively, imp = 6.82105 and dif = 6.97104,
which are significantly lower than those predicted from Eq. 8.34
and Eq. 8.38. Strauss (1975) compared the predicted values from
various correlations and found that the diffusion collection
efficiency dif from Eq. 8.36 tends to predict the lower value than
the experimental values.
c. If we choose to use c = 0.0059 which was predicted from Eqs. 8.34,
8.35, 8.38, and 8.42, the collection efficiency for the filter bed can
be estimated from Eq. 8.50 as
4B
f 1 exp
c
D c 1
410
0.03
f 1 exp
0.0059 0.79
3 1 0.03
1.5 10
Example 8.3
The collection efficiency including the interference effects is from Eq.
8.51
Example 8.3
vo
cm/s
0.1
1
5
10
15
20
50