CHE 514A: BIOCHEMICAL ENGINEERING

CHAPTER 8: STERILIZATION

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 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
The detailed description of each method is discussed in your books. Read it.

Thermal Death Kinetics

Thermal death of microorganisms at a particular temperature can be described by first order kinetics:
𝑑𝑛
= −𝑘𝑑 𝑛
𝑑𝑡
Where kd =specific death rate
The value of kd depends not only on the type of species but also on the physiological form of cells i.e. the value of k d for
bacterial spores at 121°C is of order 1 min-1, whereas those for vegetative cells vary from 10 to about 10 10 min–1
depending on the particular organism.
Upon integration yields:
𝑡
𝑛
ln = − ∫ 𝑘𝑑 𝑑𝑡
𝑛0 0
𝑡
𝑛 = 𝑛0 𝑒𝑥𝑝 (− ∫ 𝑘𝑑 𝑑𝑡)
0
𝒏 = 𝒏𝟎 𝐞𝐱𝐩(−𝒌𝒅 𝒕)
Where: n = final number of cells
n0 = initial number of cells
t = time of sterilization
Linear form:
ln 𝑛 = ln 𝑛0 − 𝑘𝑑 𝑡
Arrhenius Equation:
−𝐸𝑎
𝑘𝑑 = 𝑘𝑑0 𝑒𝑥𝑝 ( )
𝑅𝑇
Linear form:
𝐸𝑎 1
ln 𝑘𝑑 = ln 𝑘𝑑0 −
𝑅 𝑇
Where: kd = specific death rate R= gas constant
Ea = activation energy T = temperature

Design Criterion
The design criterion, ∇ for sterilization (Deindoerfer and Humphrey, 1959) is given by:
𝒕
𝒏𝟎
𝛁 = 𝐥𝐧 = ∫ 𝒌𝒅 𝒅𝒕
𝒏 𝟎

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Where ∇ is also known as Del factor, a measure of the size to be accomplished. It will give a quantitative measure of the
work that needs to be done in order to kill the necessary microorganisms. The Del factor can be solved by using the
number of microorganisms or the specific death rate.

Substituting the Arrhenius equation, Del factor can also be equal to

𝒕
−𝑬𝒂
𝛁 = 𝒌𝒅𝟎 ∫ 𝒆𝒙𝒑 ( ) 𝒅𝒕
𝟎 𝑹𝑻

n0 in the previous equation is the initial number of cells, although n is the final number of cells it is not usually expressed
as the final number but in terms of probability. This is because, of course, after sterilization as much as possible there
would be no bacteria or microorganisms left in the medium but if we are to solve for Del factor with n = 0, it would give
a Del factor equal to infinity which is technically impossible. So we represent n in terms of probability. For example, an n
= 0.001 would mean that there is a 1 in 100 chance of a bacteria surviving.

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 in exact order. Therefore, the total Del factor required should be equal to the sum of the Del factor for heating,
holding, and cooling as
∇𝑡𝑜𝑡𝑎𝑙 = ∇ℎ𝑒𝑎𝑡 + ∇ℎ𝑜𝑙𝑑 + ∇𝑐𝑜𝑜𝑙

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Shown on the figure above is a graphic representation of the three stages of sterilization. Shown on the x-axis is time
and on the y-axis is temperature. Pay attention to the linear curve of the graph only. So initially we have the initial
temperature of the medium at raw medium temperature, then during heating temperature would increase up to the
holding temperature. The holding stage is the most critical and important among the three stages. Although the amount
of time for this stage is only short, its temperature is at maximum, so majority of the microorganisms die during holding.
After holding stage it would now be followed by cooling down to the fermentation temperature, which is at normal
temperature.

In order to estimate the holding time, a design procedure is discussed as follows:

1. Calculate the total sterilization criterion by using n and n 0.

2. Calculate for the time needed during heating and cooling by making use of the following equations.
a. For batch heating by direct steam sparging into the medium:
𝐻𝑚𝑆 𝑡
𝑇 = 𝑇0 +
𝑐(𝑀 + 𝑚𝑆 𝑡)
b. For batch heating with constant rate of heat flow:
𝑞𝑇𝑡
𝑇 = 𝑇0 +
𝑐𝑀
c. For batch heating with isothermal hear source:
−𝑈𝐴𝑡
𝑇 = 𝑇𝐻 + (𝑇0 − 𝑇𝐻 )𝑒𝑥𝑝 ( )
𝑐𝑀
d. For batch cooling using a continuous nonisothermal heat sink such as passing cooling water through cooling
coils: (This formula is different from your book, kindly change what is written in the book.)
−𝑈𝐴 𝑚𝐶 𝑡
𝑇 = 𝑇𝐶0 + (𝑇0 − 𝑇𝐶0 )𝑒𝑥𝑝 {− [1 − exp ( )] }
𝑚𝐶 𝑐 𝑀
Where: T = final temperature after heating/cooling
T0 = initial temperature before heating/cooling
H = enthalpy change

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mS = mass flowrate of steam used for heating
t = time of heating/cooling
c = heat capacity of medium
M = mass of medium
q = rate of heat transfer
TH = temperature of heat source
TC0 = temperature of cooling water
U = overall heat transfer coefficient
A = heat transfer area
mC = mass flowrate of cooling water

3. Solve for ∇ℎ𝑒𝑎𝑡 and ∇𝑐𝑜𝑜𝑙 using the formula for Del factor.
4. The holding time can be calculated from
∇ℎ𝑜𝑙𝑑 ∇𝑡𝑜𝑡𝑎𝑙 − ∇ℎ𝑒𝑎𝑡 − ∇𝑐𝑜𝑜𝑙
𝑡ℎ𝑜𝑙𝑑 = =
𝑘𝑑 𝑘𝑑

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PROBLEM #1:
A fermentation medium contains an initial spores concentration of 8.5x10 10. The medium is sterilized thermally at 120oC,
and the spore density was noted with the progress of time as given below:
Time (min) 0 5 10 15 20 30
10 9 7 6 4
Spore density 8.5x10 4.23x10 6.2x10 1.8x10 4.5x10 32.5
(m-3)

a) Find the thermal specific death rate.

b) Calculate the survival factor at 40 min.

SOLUTION:
a. Since there are varying data points, namely the time with a corresponding concentration, a linear equation will
be used to solve for kd.
ln 𝑛 = ln 𝑛0 − 𝑘𝑑 𝑡
X is time which is given in the table and Y is n which is also given in terms of spore density. Substituting in the
linear equation,
𝑘𝑑 = 0.7413 𝑚𝑖𝑛−1

b. The survival factor is a ratio of how much bacteria survived after sterilization. In order to solve for it, we need
the value of n
𝑛 = 𝑛0 exp(−𝑘𝑑 𝑡)
𝑛 = (8.5𝑥1010 ) exp(−0.7413) (40)
𝑛 = 0.0113

𝑛 0.0113
𝑆𝑓 = =
𝑛0 8.5𝑥1010

𝑆𝑓 = 1.3294𝑥10−13

PROBLEM #2:
The thermal death kinetic data of Bacillus stearothermophilus(which is one of the most heat-resistant microbial type) are
as follows at three different temperatures:
Temperature (°C) 115 120 125
-1
Kd (min ) 0.035 0.112 0.347

a) Calculate the activation energy (Ed) and Arrhenius constant (kd0) of the thermal specific death rate kd.
b) Find kd at 130oC.

SOLUTION:
a. Since there are varying data points of temperature with corresponding k d, a linear equation will be used.
𝐸𝑎 1
ln 𝑘𝑑 = ln 𝑘𝑑0 −
𝑅 𝑇
The Ea can be solved from the slope and k d0 from y-intercept.

𝐽
𝐸𝑎 = 294745.9627
𝑚𝑜𝑙
𝑘𝑑0 = 1.6243𝑥1038 𝑚𝑖𝑛−1
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b. The kd at a different temperature can be solved from Arrhenius equation.

−𝐸𝑎
𝑘𝑑 = 𝑘𝑑0 𝑒𝑥𝑝 ( )
𝑅𝑇
−294745.9627
𝑘𝑑 = (1.6243𝑥1038 ) exp ( )
(8.314)(130 + 273.15)

𝑘𝑑 = 1.0475 𝑚𝑖𝑛−1

PROBLEM #3:
A culture medium that is contaminated with 1010m–3 microbial spores of microorganisms will be heat-sterilized with
steam at 121°C. At 121°C, the specific death rate of the spores can be assumed to be 3.2 min –1. When the contamination
must be reduced to one in 1000 fermentations, estimate the required sterilization time.
GIVEN:
n0 = 1010 /m3
n = 1/1000
T = 121°C
kd = 3.2/min

REQUIRED: t

SOLUTION:
The time of sterilization can be estimated from the formula for Del factor, assume a 1 m 3 volume of medium.
𝑛0
∇ = ln = 𝑘𝑑 𝑡
𝑛
1010
𝑚3 (1 𝑚3 ) 3.2
ln =( ) (𝑡)
1 𝑚𝑖𝑖𝑛
1000
𝑡 = 9.3543 𝑚𝑖𝑛

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 CHE 514A: BIOCHEMICAL ENGINEERING
PROBLEM #4:
(Ex. 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 5x10 12m–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/h, 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 proper holding time, the fermenter will
be cooled by passing 100 m3/h 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/h·m2·K. The heat-resistant bacterial spores in the medium can be characterized by an Arrhenius
coefficient (kd0) of 5.7x1039h–1 and an activation energy (Ed) of 2.834x105 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.

GIVEN:
V of medium = 40 m3 Cooling:
T0 = 25°C QC = 100 m3/h
n0 = 5x1012 m-3 TC0 = 20°C
n = 1/1000 T = 30°C
Heating: A = 40 m2
Direct injection of sat’d steam U = 2500 kJ/h.m2.K
P = 345 kPa
mS = 5000 kg/h kd0 = 5.7x1039 h-1
T = 122°C Ea = 2.834x105 kJ/kmol
c = 4.187 kJ/kg.K
ρ = 1000 kg/m3

REQUIRED: holding time

SOLUTION:
In order to solve for the holding time,
∇ℎ𝑜𝑙𝑑 ∇𝑡𝑜𝑡𝑎𝑙 − ∇ℎ𝑒𝑎𝑡 − ∇𝑐𝑜𝑜𝑙
𝑡ℎ𝑜𝑙𝑑 = =
𝑘𝑑 𝑘𝑑
The ∇𝑡𝑜𝑡𝑎𝑙 can be computed since n0 and n for the entire sterilization process is given. So,
𝑛0 (5𝑥1012 /𝑚3 )(40 𝑚3 )
∇𝑡𝑜𝑡𝑎𝑙 = ln = ln = 39.8371
𝑛 1
1000
For heating, the formula for Del factor can also be made use of. The formula in terms of number of bacteria will not
suffice since we have no idea what is n after heating. So instead we will use
𝑡 −𝐸𝑎
∇ℎ𝑒𝑎𝑡 = 𝑘𝑑0 ∫ 𝑒𝑥𝑝 ( ) 𝑑𝑡
0 𝑅𝑇
The Ea, T and kd0 is given in the problem, all we need is the time for heating. From the concept part earlier, for direct steam
injection,
𝐻𝑚𝑆 𝑡
𝑇 = 𝑇0 +
𝑐(𝑀 + 𝑚𝑆 𝑡)
From the book:
@ Psteam = 345kPa, Hv = 2731 kJ/kg (latent heat of vapour which is the steam so use the property of steam)

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@ Tmedium = 25°C, HL = 105 kJ/kg (latent heat of liquid which is the medium so use property of medium)
(Change in enthalpy) H = HV – HL = 2731 – 105 = 2626 kJ/kg

Substituting: (the final temperature after heating is the holding temperature since heating is followed by holding.)
𝑘𝐽 𝑘𝑔
(2626 ) (5000 ) (𝑡)
𝑘𝑔 ℎ
(122 + 273.15)𝐾 = (25 + 273.15)𝐾 +
𝑘𝐽 𝑘𝑔 𝑘𝑔
(4.187 ) [(40 𝑚3 ) (1000 3 ) + (5000 ) (𝑡)]
𝑘𝑔. 𝐾 𝑚 ℎ

𝑡 = 1.4637 ℎ T = this whole term

The T in the formula for Del factor is the temperature during heating. This temperature is increasing with respect to
time. Since it is changing together with time the ∇ℎ𝑒𝑎𝑡 will be computed in terms of differential change, that is taking the
integral of time.

39
1.4637 −2.834𝑥105 𝑘𝐽/𝑘𝑚𝑜𝑙
∇ℎ𝑒𝑎𝑡 = (5.7𝑥10 /ℎ) ∫ 𝑒𝑥𝑝 ( ) 𝑑𝑡
0 𝑘𝐽 2626(5000)(𝑡)
8.314 [298.15 + ]
𝑘𝑚𝑜𝑙. 𝐾 4.187(4000 + 5000𝑡)

∇ℎ𝑒𝑎𝑡 = 15.4153

For cooling, it will be computed the same way as heating but making use of the appropriate formula for cooling, (The
initial temperature T0 before cooling is the holding temperature since before cooling, holding stage occurred.)
−𝑈𝐴 𝑚𝐶 𝑡
𝑇 = 𝑇𝐶0 + (𝑇0 − 𝑇𝐶0 )𝑒𝑥𝑝 {− [1 − exp (
)] }
𝑚𝐶 𝑐 𝑀
(−2500)(40) (100)(1000)(𝑡)
(30 + 273.15) = (20 + 273.15) + (122 − 20) exp {− [1 − 𝑒𝑥𝑝 ( )] }
(100)(1000)(4.187) (40)(1000)
𝑡 = 4.3725 ℎ

𝑡
−𝐸𝑎
∇𝑐𝑜𝑜𝑙 = 𝑘𝑑0 ∫ 𝑒𝑥𝑝 ( ) 𝑑𝑡
0 𝑅𝑇
∇𝑐𝑜𝑜𝑙 =
4.3725
−2.834𝑥105 𝑘𝐽/𝑘𝑚𝑜𝑙
= (5.7𝑥1039 ) ∫ 𝑒𝑥𝑝 ( ) 𝑑𝑡
0 𝑘𝐽 −2500(40) 100,000𝑡
8.314 [293.15 + (102 exp {− [1 − 𝑒𝑥𝑝 ( )] }}
𝑘𝑚𝑜𝑙. 𝐾 100(1000)(4.187) 40,000

∇𝑐𝑜𝑜𝑙 = 16.9560

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All we need now to solve for holding time is k d by using Arrhenius equation. (The temperature to be used in solving for kd is the
holding temperature since the kd we need is for holding.)
−2.834𝑥105 𝑘𝐽/𝑘𝑚𝑜𝑙
𝑘𝑑 = (5.7𝑥1039 /ℎ) 𝑒𝑥𝑝 ( ) = 195.9056 ℎ−1
8.314(122 + 273.15)
So,
39.8371 − 15.4153 − 16.9560
𝑡ℎ𝑜𝑙𝑑 =
195.9056
𝑡ℎ𝑜𝑙𝑑 = 0.0381 ℎ = 2.286 𝑚𝑖𝑛

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