Sterilization_2022_Part 1

Sterilization
Contents:
- Death kinetics
- Thermal sterilization of liquid medium
- Air sterilization
- Equations describing sterilization
operations

Prof. R. Shanthini
1
Sterilization is a process to kill or inactivate
all viable organisms in a culture medium or
in a gas or in the fermentation equipment.
A reactor is built to promote cell growth, but inputs as well as
outputs from the reactor must be devoid of any organism.

This is, however, not possible in practice to kill or inactivate all

viable organisms.

Commercial sterilization is therefore aims at reduce risk of

contamination to an acceptable level.

Factors determining the degree of sterilization include safety,

cost and effect on product.
Prof. R. Shanthini 2
Reasons for Sterilization:
• Many fermentations must be absolutely devoid of foreign organisms.
• Otherwise production organism must compete with the foreign
organisms (contaminants) for nutrients.
• Foreign organisms can produce harmful (or unwanted) products
which may inhibit the growth of the production organisms.
• Economic penalty is high for loss of sterility.
• Recombinant DNA fermentations - exit streams must be sterilized.
and more….

Recombinant DNA (rDNA) molecules are DNA molecules formed by

laboratory methods of genetic recombination that bring together genetic
material from multiple sources, creating sequences that would not
otherwise be found in the genome (which is the complete set of genetic
information in an organism).
Prof. R. Shanthini 3
Sterilization methods:
• Thermal:
preferred for economical large-scale sterilizations of liquids and equipment

• Chemical:
preferred for heat-sensitive equipment
→ ethylene oxide (gas) for equipment
→ 70% ethanol-water (pH=2) for equipment/surfaces
→ 3% sodium hypochlorite for equipment

• Irradiation:
→ ultraviolet for surfaces
→ X-rays for liquids (costly/safety)

Prof. R. Shanthini 4
Sterilization methods continued:
• Sonication (sonic / ultrasonic vibrations)

• High-speed centrifugation

• Filtration (preferred for heat-sensitive material and filtered air)

Prof. R. Shanthini 5
Thermal Sterilization:
Dry air or super-heated steam
can be used as the heat agent.
Heat kills microorganisms by
denaturing their enzymes
and other proteins. Heat
resistance varies widely among
microbes.
Moist (wet) steam can also be used
as the heat agent (eg: done at 121oC
at 2 bar).
Moist heat kills microorganisms by
coagulating their proteins.
Death rate of moist cells are higher
than that of the dry cells since
moisture conducts heat better than a
dry system. Therefore, moist steam
is more effective than dry air/steam.

Thermal sterilization does not contaminate the medium

of equipment that was sterilized (as in the case of use of
chemical agent for sterilization).
Prof. R. Shanthini 6
Thermal sterilization using dry heat:
- Direct flaming

- Incineration
medical wastes are burned to produce
combustion gases and ashes

- Hot air oven

at 170oC for 1 hour
at 140oC for 3 hours

Prof. R. Shanthini 7
Thermal sterilization using moist heat:
- Pasteurization (below 100oC)
Destroys pathogens without altering the flavor of the food.
Classic method: 63oC; 30 min
High Temperature/Short Time (HTST) : 71.7oC; 15-20 sec
Untra High Temperature (UHT) : 135oC; 1 sec
- Boiling (at 100oC)
killing most vegetative forms microorganisms
Requires 10 min or longer time
Hepatitis virus can survive for 30 min & endospores for 20 h
- Autoclaving (above 100oC)
killing both vegetative organisms and endospores
121-132oC; 15 min or longer

Prof. R. Shanthini 8
Autoclave:
It is done in a chamber which
is filled with hot steam under
pressure.

Preferred method of
sterilization, unless material is
damaged by heat, moisture,
or high pressure.

Prof. R. Shanthini 9
Spores: Hyphal growth

Spore Spore
germination production

Spores

Spores form part of the life cycles of many bacteria, plants, algae, fungi and some
protozoa. (There are viable bacterial spores that have been found that are 40 million
years old on Earth and they're very hardened to radiation.) A spore is a reproductive
structure that is adapted for dispersal and surviving for extended periods of time in
unfavorable conditions. A chief difference between spores and seeds as dispersal
units is that spores have very little stored food resources compared with seeds.
Prof. R. Shanthini 10
Endospores:
Spore and endospore are two resistant structures living organisms produce.
Both are capable of giving rise to a new individual.

The main difference between spore and endospore is that spore is an

active reproductive structure mainly produced by plants and fungi
whereas endospore is a dormant, non-reproductive structure of
bacteria.

Source: https://pediaa.com/what-is-the-difference-between-spore-and-endospore/

Prof. R. Shanthini 11
Thermal Cell Death Kinetics (First-order Model):

(S.1)

where
is the cell death rate
is the first-order thermal specific death rate
is the concentration (or number) of live organisms present

Prof. R. Shanthini 12
Thermal Cell Death Kinetics (First-order Model):

(S.1)

where
is the cell death rate
is the first-order thermal specific death rate
is the concentration (or number) of live organisms present

Compare (S.1) to cell growth kinetics that we have already learned.

where
is the cell growth rate
is the first-order thermal specific growth rate

Prof. R. Shanthini 13
Thermal Cell Death Kinetics (First-order Model):

(S.1)

depends on the type of species,

the physiological form of the cells, and
the temperature.

for vegetative cells >>> for spores > for virus

at high temperature > at low temperature

Prof. R. Shanthini 14
Thermal Cell Death Kinetics (First-order Model):

(S.1)

It is common to consider number of cells instead of concentration

of cells since number of cells is easily estimated under microscope.
Therefore, we will replace by (which is the number of live
organisms present at time ).

(S.1) can, therefore, be written as follows:

(S.2)

Unit of is number of cells per unit volume (or unit area) per unit time.

Prof. R. Shanthini 15
Thermal death kinetics in a batch reactor: 𝑟 = −𝑘 𝑛 (S.2)

Cell mass balance over a batch reactor gives

(S.3)
Combining (S.2) and (S.3), we get
(S.4)
where
is the number of live organisms present at time
is the sterilization time
is the first-order thermal specific death rate
Integrating (S.4) using the initial condition = at = 0 gives

(S.5)
Prof. R. Shanthini 16
Thermal Death Kinetics ln
𝑛
=− 𝑘 𝑑𝑡 (S.5)
𝑛
(isothermal operation):
is a function of temperature, and is a constant for isothermal operations.
(S.5) therefore gives
(S.6)

Survival factor
(S.7)

1
Inactivation factor ≡ = =
Survival factor

Prof. R. Shanthini 17
Exercise 1:

A fermentation medium contains an initial spores concentration of 8.5 x 1010. The

medium is sterilized isothermally at 120oC, and the spore density was noted with the
progress of time as given below:
Time 0 5 10 15 20 30
(min)
Spore density 8.5 x 1010 4.23 x 109 6.2 x 107 1.8 x 106 4.5 x 104 32.5
(m-3)

a) Find the thermal specific death rate.

b) Plot the survival factor against time at 120oC and comment on its shape.
c) Determine the number of live organisms at 40 min and comment on your result.

Prof. R. Shanthini 18
Solution to Exercise 1:

Data provided: = 8.5 x 1010

versus data are given
Isothermal operation at 120oC.

a) Find the thermal specific death rate ( ).

Since it is an isothermal operation, is a constant. Therefore, (S.6) can be used

as follows:

(S.6)

Plotting versus and finding the slope will give the numerical value of

Prof. R. Shanthini 19
 0
0 10 20 30 40

-5

-10

-15
y = -0.7201x
R² = 0.9988
-20

-25

Prof. R. Shanthini 20
b) Plot the survival factor against time at 120oC and comment on its shape
At 120oC, survival factor is given by (S.4) as follows:

0.8
Survival factor

0.6

0.4

0.2

0
0 5 10 15 20 25
Prof. R. Shanthini min) 21
c) Calculate the number of live organisms at 40 min and comment on your result.

The survival factor at 40 min can be calculated as follows:

Since , =

Can be lower than 1? NO….

So we interpret the above number as follows:
The chances of survival for the living organism is 26 in 1000.

Prof. R. Shanthini 22
Thermal Death Kinetics
(non-isothermal operation):
Temperature dependence of is expressed by the Arrhenius equation as below:

(S.8)

where
is the Arrhenius constant for thermal cell death
is the activation energy for thermal cell death
is the universal gas constant
is the absolute temperature

Prof. R. Shanthini 23
Thermal Death Kinetics 𝑘 = 𝑘 exp −
𝐸
(S.8)
𝑅𝑇
(non-isothermal operation):
𝑛
ln =− 𝑘 𝑑𝑡 (S.5)
𝑛
When of (S.8) is substituted in (S.5), we get

(S.9)

To carry out the above integration, we need to know how the temperature
( ) changes with time ( ).

We get that information from the experimental data or from writing energy
balances over the system considered.

Prof. R. Shanthini 24
 𝐸
Determining the Arrhenius constants: 𝑘 = 𝑘 exp −
𝑅𝑇
(S.8)

Taking natural logarithm of of (S.8), we get

(S.10)

Prof. R. Shanthini 25
Exercise 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 (oC) 115 120 125

kd (min-1) 0.035 0.112 0.347

a) Calculate the activation energy ( ) and Arrhenius constant ( ) of the thermal

specific death rate .
b) Find at 130oC.
c) Plot the survival factor of Bacillus stearothermophilus against time at 115, 120,
125 and 130oC
Answers: a) 𝐸 = 294.5 kJ/mol; 𝑘 = 2.616 x 1036 per s;
b) 𝑘 at 130oC is 1.054 per min
Prof. R. Shanthini 26
Solution to Exercise 2:

a) Calculate the activation energy ( ) and Arrhenius constant ( ) of the thermal

specific death rate .
Answer: 294.5 kJ/mol; 2.616 x 1036 per s;

b) Find at 130oC.
Answer: at 130oC is 1.054 per min

c) Plot the survival factor of Bacillus stearothermophilus against time at 115, 120,
125 and 130oC

At isothermal condition, survival factor is given by (S.7) as follows:

(S.7)
Prof. R. Shanthini 27
 1
at 115 degC
0.9
at 120 degC
0.8
at 125 degC
0.7
Survival factor

at 130 degC
0.6

0.5

0.4

0.3

0.2

0.1

0
0 20 40 60 80 100 120 140
Prof. R. Shanthini min) 28
Design Criterion ln
𝑛
=− 𝑘 𝑑𝑡 (S.5)
𝑛
for Sterilization:
𝑛 𝐸
ln = −𝑘 exp − 𝑑𝑡 (S.9)
𝑛 𝑅𝑇

 (S.11)

Del factor (which is a measure of fractional

reduction in living organisms count over the
initial number present)

Prof. R. Shanthini 29
Determine the Del factor to reduce the number of cells in a fermenter from 1010
viable organisms to 1.

Even if one organism is left alive, the whole fermenter may be contaminated.

Therefore, no organism must be left alive. That is, should be 0.

To achieve this del factor, we need infinite time that is not possible.

Therefore should not be 1, and it cannot be 0.

Prof. R. Shanthini 30
Let is choose = 0.001
(It means the chances of survival for the living organism is 1 in 1000.)


.

Using Arrhenius Law, we get

Temperature profile during sterilization must be

chosen such that the Del factor can become 30.

Prof. R. Shanthini 31
Typical temperature profile during sterilization:
heating
holding

cooling

Prof. R. Shanthini 32