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James M. Lee
Department of Chemical Engineering
Washington State University
Pullman, WA 99164-2714
jmlee@wsu.edu
Chapter 8 Sterilization.................................... 1
8.1. Sterilization Methods.................................................................... 1
8.2. Thermal Death Kinetics ................................................................ 2
8.3. Design Criterion............................................................................ 3
8.4. Batch Sterilization......................................................................... 4
8.5. Continuous Sterilization ............................................................... 7
8.6. Air Sterilization........................................................................... 13
8.7. Nomenclature .............................................................................. 22
8.8. Problems...................................................................................... 24
8.9. References................................................................................... 26
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 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.
8.1. Sterilization Methods
Sterilization of fermentation media or equipment can be accomplished by
destroying all living organisms by means of heat (moist or dry), chemical
agents, radiation (ultraviolet or X-rays), and mechanical means (sonic or
ultrasonic vibrations). Another approach is to remove the living organisms by
means of filtration or high-speed centrifugation.
Heat is the most widely used means of sterilization, which can be
employed for both liquid medium and heatable solid objects. It can be applied
as dry or moist heat (steam). The moist heat is more effective than the dry
heat, because the intrinsic heat resistance of vegetative bacterial cells is
greatly increased in a completely dry state. As a result the death rate is much
lower for the dry cells than for moist ones. The heat conduction in dry air is
also less rapid than in steam. Therefore, dry heat is used only for the
sterilization of glassware or heatable solid materials. By pressurizing a
vessel, the steam temperature can be increased significantly above the boiling
point of water. Laboratory autoclaves are commonly operated at a steam
pressure of about 30 psia, which corresponds to 121°C. Even bacterial spores
are rapidly killed at 121°C.
8-2 Sterilization
dn
= −kd n (8.1)
dt
where kd is specific death rate, the value of which depends not only on the
type of species but also on the physiological form of cells. For example, the
value of kd for bacterial spores at 121°C is of the order of 1 min−1, whereas
those for vegetative cells vary from 10 to about 1010 min–1 depending on the
particular organism (Aiba et al., 1973).
Integration of Eq. (8.1) yields
n t
ln = −∫ kddt (8.2)
n0 0
or
( )
t
n = n 0 exp −∫ kddt (8.3)
0
which shows the exponential decay of the cell population. The temperature
dependence of the specific death rate kd can be assumed to follow the
Arrhenius equation:
kd = kd0 exp − d
E
RT
( ) (8.4)
where Ed is activation energy, which can be obtained from the slope of the
ln(kd) versus 1/T plot. For example, the activation energy of E. coli is 127
kcal/gmole and that of Bacillus stearothermophilus Fs 7954 is 68.7 (Aiba et
al., 1973).
8.3. Design Criterion
From Eqs. (8.2) and (8.4), the design criterion for sterilization ∇ can be
defined as (Deindoerfer and Humphrey, 1959)
n E
( )
t t
∇ = ln 0 = ∫ kddt = kd0 ∫ exp − d dt (8.5)
n 0 0 RT
which is also known as the Del factor, a measure of the size of the job to be
accomplished. The Del factor increases as the final number of cells decreases.
For example, the Del factor to reduce the number of cells in a fermenter from
1010 viable organisms to one is
1010
∇ = ln = 23 (8.6)
1
The reduction of the number of cells from 1010 to one seems to be
impressive. However, even if one organism is left alive, the whole fermenter
8-4 Sterilization
Hmst
T = T0 + (8.9)
c ( M + mst )
b. For batch heating with a constant rate of heat flow such as electrical
heating, the linear form is used:
qTt
T = T0 + (8.10)
cM
c. For batch heating with a isothermal heat source such as steam
circulation through heating coil, the exponential form is used:
T = TH + (T0 − TH ) −
UAt
(8.11)
cM
d. For batch cooling using a continuous nonisothermal heat sink such
as passing cooling water through cooling coil, the exponential form
is used:
UA mct
T = TC0 + (T0 − TC0 ) exp − 1 − exp − M (8.12)
m c
c
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. If using
theoretical equations, integrate Eq. (8.5) numerically after substituting
in the proper temperature profiles. Then, the holding time can be
calculated from
∇ ∇ + ∇ hold + ∇ cool
thold = total = heat (8.13)
kd kd
––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
Example 8.1
A fermenter containing 40 m3 of medium (25°C) is going to be sterilized by
the direct injection of saturated steam. The typical bacterial count of the
medium is about 5×1012 m−3, 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/hr, which will be stopped when the medium temperature reaches 122°C.
During the holding time, the heat loss through the vessel is assumed to be
negligible. After a proper holding time, the fermenter will be cooled by
8-6 Sterilization
passing 100 m3/hr of 20°C water through the cooling coil in the fermenter
until the medium reaches 30°C. The coil has a heat-transfer area of 40 m2 and
for this operation the average overall heat-transfer coefficient (U) for cooling
is 2,500 kJ/hr m2 K. The heat-resistant bacterial spores in the medium can be
characterized by an Arrhenius coefficient kd0 of 5.7×1039 hr–1 and an
activation energy (Ed) of 2.834×105 kJ/kmol (Deindoerfer and Humphrey,
1959). The heat capacity and density of the medium are 4.187 kJ/kg K and
1,000 kg/m3, respectively. Estimate the required holding time.
Solution:
The design criterion can be calculated from Eq. (8.5) as
n ( )( )
∇ = ln 0 = ln 5 × 10 m −3 40m = 39.8
12 -3 3
n 1 × 10
The direct injection of steam into the medium can be assumed to follow
the hyperbolic temperature-time profile of Eq. (8.9), which can be used to
calculate the time required to heat the medium from 25°C to 122°C. From the
steam table (Felder and Rousseau, 1986), the enthalpy of saturated steam at
345 kPa and water at 25°C is 2,731 and 105 kJ/kg, respectively. Therefore,
the enthalpy of the saturated steam at 345 kPa relative to raw medium
temperature (25°C) is
H = 2,731 − 105 = 2,626 kJ/kg
From Eq. (8.9),
(2,626 kJ/kg)(5,000 kg/hr)t 78.4t
T = T0 + 3 3
= T0 +
(4.187 kJ/kg K)[(40 m )(1,000 kg/m ) + (5,000 kg/hr)t ] 1 + 0.125t
steam injector heats the medium to the peak sterilization temperature quickly.
Therefore, sterilization during the heating period is negligible.
For indirect heating, the plate-and-frame heat exchanger is generally more
effective than the shell-and-tube type for heat transfer due to its larger heat-
transfer area. However, the former is limited to lower pressures (normally
less than 20 atm) due to its weak structural strength compared with the latter.
The plate-and-frame type is also favorable for the sterilization of a high
viscous system.
The temperature change with respect to residence time (τ hold ) as the
medium passes through an isothermal heat source can be approximated as
(Deindoerfer and Humphrey, 1959b),
UAτ heat
TC2 = TH − (TH − TC1 )exp − (8.14)
cW
For heating using a countercurrent heat source of equal flow rate and heat
capacity,
∆TUAτ heat
TC2 = TC1 − (8.15)
cW
Holding Section: The heated medium passes through a holding section,
which is usually composed of a long tube. The holding section is maintained
in adiabatic conditions. If the heat loss in the section is negligible, the
temperature can be assumed to be constant. The average residence time in the
holding section is
L
τ hold = (8.16)
u
from which the Del factor can be estimated as
n E
∇hold = ln 0 = kdτ hold = kd0 exp − d τ hold (8.17)
n RT
the pipe wall. For laminar flow of Newtonian fluids through a smooth round
pipe, the ratio of the average fluid velocity to the maximum velocity /line
u/um is 0.5. The ratio changes rapidly from 0.5 to about 0.75 when laminar
flow changes to turbulent, and then increases gradually to 0.87 when the
Reynolds number is about 106 (McCabe et al., 1985). As a result, if we use
the mean velocity in calculating the required residence time for sterilization,
some portion of medium will be understerilized, which may cause a serious
contamination problem.
.............
.............
.............
.............
.............
...................................................... ............. ......................................................
d(uCn )
Bulk flow uCn S ............. uCn S + dxS
.............
.............
dx
. ..... .
...................
dCn .............
dCn d dCn
Dispersion −DS ...................................................... .................... ......................................................
...................
−DS + −DS dx
dx ............. dx dx dx
.............
.............
.......................................
dx
Figure 8.1 Material balances around an elementary section in a holding
tube.
The deviation from ideal plug flow due to the axial mixing can be
described by the dispersion model (Levenspiel, 1972). Let's look at the
differential element with a thickness dx in a holding tube as shown in Figure
8.1. The basic material balance for the microorganisms suspended in the
medium is
In - Out -Killed by Sterilization = Accumulation (8.18)
At steady state, the accumulation term is equal to zero. The input and output
of the microorganisms into or out of the element have both a bulk flow and
an axial diffusion condition. The number of microorganisms entering minus
those leaving by bulk flow is
d (uCn )
uCn S − uCn S + Sdx (8.19)
dx
Analogous to the molecular diffusion, the x-directional flux of
microorganisms suspended in a medium due to the axial mixing can be
represented as
dC
J n = −D n (8.20)
dx
where D is the axial dispersion coefficient, characterized by the degree of
backmixing during flow. The mechanism of axial dispersion may be
molecular or turbulent. If D is zero, the velocity distribution approaches that
of the ideal plug flow. At the other extreme, if D is infinitely large, the fluid
8-10 Sterilization
10.0
D
1.0
udt
0.1
1.0E+03 1.0E+04 1.0E+05 1.0E+06
dt u ρ
N Re =
µL
in the pipe is well mixed like a fully mixed vessel. For the turbulent flow, the
dispersion coefficient is correlated as a function of Reynolds number as
shown in Figure 8.2.
The number of microorganisms entering and leaving by axial dispersion is
dC dC d
− DS n − − DS n + − DS n dx
dC
(8.21)
dx dx dx dx
The number of cells killed by sterilization is kdCnSdx. Therefore, by
substituting Eqs. (8.19) and (8.21) into Eq. (8.18) and simplifying,
d dCn d ( uCn )
D − − k d Cn = 0 (8.22)
dx dx dx
For the constant D and u , Eq. (8.22) can be modified into a dimensionless
form,
d 2Cn′ dCn′ k L
− N − N Pe d Cn′ = 0 (8.23)
dx′ dx′
2 Pe
u
where
Sterilization 8-11
Cn x uL
Cn′ = x′ = N Pe =
Cn0 L D
NPe is known as Péclet number. When NPe = ∞, it is ideal plug flow. The
boundary conditions for the solution of Eq. (8.23) are
dCn′
+ N Pe (1 − Cn′ ) = 0 at x′ = 0
dx′
(8.24)
dCn′
=0 at x′ = 1
dx′
The solution of Eq. (8.23) (Wehner and Wilhelm, 1956) is
4ϕ exp Pe
N
Cn′ x′=1 = 2 (8.25)
(1 + ϕ ) exp ϕ N Pe − (1 − ϕ ) exp − ϕ N Pe
2 2
2 2
where
4k d L / u
ϕ = 1+ (8.26)
N Pe
––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
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/hr. The
time for heating and cooling is negligible with this type of sterilizer. The
typical bacterial count of the medium is about 5×1012 m−3, which needs to be
reduced to such an extent that only one organism can survive during two
months of continuous operation. The heat-resistant bacterial spores in the
medium can be characterized by an Arrhenius coefficient (kd0) of 5.7×1039
hr–1 and an activation energy (Ed) of 2.834×105 kJ/kmol (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 125°C. The physical
properties of this medium at 125°C are c = 4.187 kJ/kg K, ρ = 1000 kg/m3,
and µ = 4 kg/m hr.
a. What length should the pipe be in the sterilizer if you assume ideal
plug flow?
8-12 Sterilization
b. What length should the pipe be in the sterilizer if the effect of axial
dispersion is considered?
Solution:
a. The design criterion can be calculated from Eq. (8.5) as
n
∇ = ln 0 = ln ( 5 × 1012 m −3 )( 2 m3 /hr ) ( 24 hr/day )( 60 days ) = 37.2
n
Since the temperature at the holding section is constant, Eq. (8.5) is
simplified to
∇ = kdτ hold
From the given data, kd can be calculated by using Eq. (8.4) to yield
kd = 378.6 hr −1
Therefore,
∇ 37.2
τ hold = = = 0.0983 hr
kd 378.6
The velocity of medium is
2 m3 / hr
u= = 245 m/hr
π 2 2
0.102 m
4
The length of the sterilizer is
L = uτ hold = 24.1 m
b. The Reynolds number for the medium flow is
0.102 (245)(1000)
N Re = = 6.24 × 103
4
From Figure 8.2
D
≈ 0.8 for N Re = 6.24 × 103
udt
Therefore,
D ≈ 0.8udt = 20 m 2 /h
Now, substituting all the values given and calculated in this problem to
Eq. (8.25) will result in an equation with only one unknown, L, which
can be solved by using any non-linear equation solver:
Sterilization 8-13
L = 26.8
Therefore, the holding section should be 26.8 m, which is 2.7 m longer
than the result from the assumption of ideal plug flow.
––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
Cooling Section: For the cooling section, a quench cooler with adequate
heat removal capacity is effective. Another technique is to inject the hot
medium through an expansion valve into a vacuum chamber, which is known
as flash cooling. Both of these take a very short time; therefore, the
sterilization during the cooling period can be assumed to be negligible.
A shell-and-tube or a plate-and-frame heat exchanger can also be
employed for cooling. The temperature versus residence time relationship for
cooling using an isothermal heat sink is
UAτ cool
TH 2 = TC − (TC − TH1 )exp − (8.27)
cW
For cooling using a countercurrent heat sink of equal flow rate and heat
capacity,
∆TUAτcool
TH 2 = TH1 − (8.28)
cW
8.6. Air Sterilization
For aerobic fermentations, air needs to be supplied continuously. Typical
aeration rates for aerobic fermentation are 0.5−1.0 vvm (air volume per liquid
volume per minute). This requires an enormous amount of air. Therefore, not
only the medium but also the air must be free of microbial contaminants. All
of the sterilization techniques discussed for medium can also be employed for
air. However, 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.
The cotton plug, routinely used as a closure for tubes or flasks of sterile
solution, is a good example of removal of microorganisms from air by a
fibrous filter. A simple air filter can be made by packing cotton into a
column. However, with cotton filters the pressure drop is high and wetting
can be a breeding ground for the contamination. Therefore, glass fibers are
favorable as filter medium because they give a lower pressure drop and are
less liable to wetting or combustion. Modern fibrous filter systems are
8-14 Sterilization
Figure 8.3 Flow pattern around cylindrical fiber, showing the path of
particles collected by inertial impaction.
Sterilization 8-15
Figure 8.4 Flow pattern around cylindrical fiber, showing the interception
collection mechanism.
8-16 Sterilization
collected more than once, which does not make sense. A better approach is to
use the following correlation,
ηc = 1 − (1 − ηimp ) (1 − ηint )(1 − ηdif ) (8.41)
which allows only the particles not collected by one mechanism to be
collected by the others. Substitution of Eqs. (8.32), (8.34), and (8.35) into
Eq. (8.41) will result in the correlation for the collection efficiency by the
combined mechanisms. Pasceri and Friedlander (1960) correlated the
combined collection efficiency as
6
ηc = 2/3 0.5 + 3κ 2 N Re
0.5
(8.42)
NSc N Rec c
As mentioned earlier, with an increase of the superficial air velocity (v0), ηimp
and ηint increase whereas ηdif decreases. Therefore, the combined collection
efficiency normally decreases to reach a minimum point and then increases
with increasing superficial air velocity.
Effect of Multiple Layers and Packing: All correlations for the collec-
tion efficiency discussed so far are based on the ideal case of a single
cylindrical collector. Now, let's examine a filter unit consisting of randomly
oriented multiple layers. Consider an area (A) of filter at a right angle to the
gas flow and with a depth dh. If the packing density α is defined as the
volume of fiber per unit volume of filter bed, the velocity within the filter
void space is equal to
v0
v= (8.43)
(1 − α )
d n 0
Cv
v
Cn 0 A (1 − α ) v 1 − α dh A (1 − α )
1−α Cn 0 +
1−α dh
dh
Cnv0
v0 d A(
Cn A (1 − α ) − v0 1 − α dh 1−α )
1−α Cn +
1−α dh (8.45)
v
= 0 Cn ( Adh)ηc Dc L
1− a
where L is the length of cylindrical fiber per unit volume of filter bed, which
is related to the packing density α and the average collector diameter Dc as
π Dc2 L
α= (8.46)
4
Simplifying Eq. (8.45) and substituting Eq. (8.46) for L gives
dC 4αηc
− n= dh (8.47)
Cn π Dc (1 − α )
which, on integration, results
Cn 4B α
ln =− η (8.48)
Cn0 π Dc 1 − α c
where B is the filter depth. Therefore, the collection efficiency for the filter
bed can be estimated as
C 4B α
η f = 1 − n = 1 − exp − ηc (8.49)
Cn0 π Dc 1 − α
When fibers are packed together in a filter bed, the velocity will be
increased and the flow pattern will be changed, which increases the
collection efficiency from impaction and interception. Chen (1955) has
determined fiber interference effects experimentally and suggests
ηα = η f (1 + 4.5α ) (8.50)
2λ d
Cf =1+ 1.257 + 0.400exp −1.10 p = 1.16
dp 2λ
The Stokes number is
C f ρ p d p2v 1.16(1) (1 × 10−4 ) (10.3)
2
NSt = = = 0.0247
18µ Dc 18 (1.80 × 10−4 )(1.5 × 10−3 )
Therefore, from Eq. (8.33), the single fiber collection efficiency by
inertial impaction is
ηimp = 0.075 NSt1.2 = 0.075 ( 0.0247 )1.2 = 8.82 × 10−4
The single fiber collection efficiency by interception is from Eq. (8.34)
for κ = d p Dc = 0.0667 and N Rec = 0.103 ,
1 κ (2 + κ )
ηint = (1 + κ ) ln (1 + κ ) − = 0.96 × 10−4
2.002 − ln N Rec 2 (1 + κ )
Therefore,
6
ηc = + 3 ( 0.0667 )2 ( 0.103)0.5 = 0.0071
( 5.41× 105 ) ( 0.103)
2/3 0.5