BIOCHEMICAL

ENGINEERING
ECH 3201

CHAPTER 3:
STERILIZATION
 Introduction
Sterility:
The absence of detectable levels of viable organisms
in a culture medium or in a gas

One of the most important operations which

differentiates a biochemical process from a
chemical process

To provide contamination free

environment (Aiba et al., 1965)

Aiba, S., Humphrey, A.E., and Millis, N.F. (1965),

Biochemical Engineering, Academic Press, New York, P.
85.
 STERILIZER
TOP LOADING
AUTOCLAVE

BENCH SCALE AUTOCLAVE

 EFFECT OF CONTAMINANT
1. Medium would be consumed unnecessarily to
support the growth of contaminating organisms
2. Contaminated product may outweight the
desired product
3. May contaminate the final product
4. The contaminated product may interfere with the
recovery of the desired product
5. Unsterile air in aerobic fermentation – spoilage
the fermentation product
 STERILIZATION OF THE MEDIUM
• To avoid contaminating organisms which
may:
1. Use the nutrient in the medium
2. Change the chemical structure of the medium
3. Change the pH
4. Create more foam
5. Produce other products
6. Convert, degrade or destroy desired product
EFFECT OF STERILIZATION ON QUALITY
OF NUTRIENTS
Interaction between NUTRIENT
COMPONENTS
– discolouration of media
– reducing sugars have carboxyl
groupings - react with amino groups of
amino acids, etc.
– carbohydrate components – separated
from the remainder medium, sterilized
separately

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 9. Maintaining 1. 2. Employing pure
optimum pH which STERILIZATION
inoculum
• Text
OF MEDIUM
discourages the
growth of undesired
organisms
3.
Sterilization
of fermenter
8. Maintaining METHOD TO
aseptic conditions in AVOID
the fermenters CONTAMINATION
during fermentation IN 4. Sterilizing
FERMENTATION the pipes,
PROCESS valves, bends
7. Disinfecting the
fermenter with a 5. Sterilization of all
non-toxic materials to be
disinfectant added to the
fermenter
6. Sterilization
of air
AGENTS FOR STERILIZATION
HEAT/THERMAL
– preferred for economical large-scale sterilizations of
liquids and equipment
– Most thermal sterilizations are at 121oC
CHEMICAL
– preferred for heat-sensitive equipment
• Ethylene oxide (gas) for equipment
• 70% ethanol-water (pH=2) for equipment/surfaces
• 3% sodium hypochlorite for equipment
RADIATION
– UV for surfaces, x-rays for liquids (costly/safety)
FILTRATION
– Membrane filters having uniform micropores
– Depth filters of glass wool
 Normal sterilization – heat treatment
1. Boiling in water
2. Passing live steam
3. Autoclaving (subjecting the medium to
steam under pressure in a pressure vessel)

• Batch or continuous process

• HTST process (high temperature, short
time)
Crude and Defined medium sterilization
• Defined media do not require much effort
compared to Crude media
• Defined media require a small amount of heating
• Crude media: may contain heat-resistant bacterial
spores, require prolonged heating
• Excessive heating – denature the proteins,
caramalize sugars, thermal degradation of
components in the nutrient medium by inter-
reaction
• Enzymes and vitamins – separated initially by
passing through bacteriological filter, added
back after sterilization of the medium.
STERILIZATION OF
LIQUID MEDIA
The liquid media which contains all essential
nutrients for cell growth is:

First heat sterilized with steam, then

Cooled down before introduction into the

bioreactor vessel
2 types of sterilization:

Batch sterilization
Continuous sterilization
 BATCH STERILIZATION
• All the contents are loaded into the sterilizer
• Steam is injected – sterilization takes place
• Contents are discharged for further processing @ transfer into
fermenter
DISADVANTAGE
• Less successful in avoiding the risk of destruction of nutrients
while destroying the contaminants as compared to the
continuous.
ADVANTAGES
1. COST- investment is lower
2. CONTAMINATION – after sterilization is over are less, str.
can be done in the same vessel, in the fermenter itself
3. CONTROL – manually, less mechanical failure

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 BATCH STERILIZATION

FIG. 1 Types of equipment for batch sterilization of media. [Adopted from S. Aiba, A.E.
Humphrey and N.F. Millis. “Media Sterilization”. In Biochemical Engineering, 2nd Ed.,
Academic Press, Inc., New York (1973) 254].
 CONTINUOUS STERILIZATION

Two types of continuous sterilization:

● Direct steam injection sterilizer

Plate heat exchanger sterilizer

 CONTINUOUS STERILIZATION
1) Direct steam injection

• Steam is directly injected along

with the medium continuously
• The heating time and heating
section are negligible as shown in
Fig 2.
• The holding time is based on the
length of holding pipe
• Sterilization takes place during
the holding period
• The steam and sterilized medium
under pressure and passed
FIG. 2 Direct steam injection type of through the expansion valve into
continuous sterilization of liquid media. the vacuum chamber
[Adopted from S. Aiba, A.E. Humphrey and • Steam is removed out under
N.F. Millis. “Media Sterilization”. In vacuum
Biochemical Engineering, 2nd Ed., Academic • the sterile medium passes
Press, Inc., New York (1973) 257]. through the cooling zone
 CONTINUOUS STERILIZATION
1) Direct steam injection
Fig. 2 temperature-time profiles for continuous sterilization.

FIG. 2. Sterilization temperature vs. time profile for direct steam injection continuous
sterilizer. [Adopted from S. Aiba, A.E. Humphrey and N.F. Millis. “Media Sterilization”. In
Biochemical Engineering, 2nd Ed., Academic Press, Inc., New York (1973) 258].
 CONTINUOUS STERILIZATION
2) Plate heat exchanger

• It is heated with steam in a heat

exchanger
• Then passed through the holding
section
• The residence time in the
holding section is decided by;
1. holding time requirement,
2. flow rate of the heated
medium
3. length of the holding
FIG. 3 Plate heat exchanger type of continuous section
sterilization of liquid media. [Adopted from S.
Aiba, A.E. Humphrey and N.F. Millis. “Media
Sterilization”. In Biochemical Engineering, 2nd
Ed., Academic Press, Inc., New York (1973) 257].
 CONTINUOUS STERILIZATION
Plate heat exchanger
Fig. 5 temperature-time profiles for continuous sterilization

FIG. 5 Sterilization temperature vs. time profile for plate heat exchanger sterilizer. [Adopted
from S. Aiba, A.E. Humphrey and N.F. Millis. “Media Sterilization”. In Biochemical
Engineering, 2nd Ed., Academic Press, Inc., New York (1973) 257].
KINETICS OF THERMAL DEATH OF
MICROORGANISMS
Practical considerations:
• Not all organisms have identical death kinetics
- (increasingly difficulty; vegetative cells < spores <
virus)
• Individuals within a population of the same organism may
respond differently
Heat is used to kill:
Contaminant microorganisms
Spores
present in a liquid nutrient medium.
The destruction of microorganisms by heat
means –
Loss of Viability of these microorganisms
and spores.
The thermal death of microorganisms follow first order kinetics given by
Eq. 1
-dN/dt = kN……………………...(1)
Where:
N = Number of viable microorganisms present
t = Sterilization time, min
k = Thermal death rate constant, min-1
The –ve sign indicates that as t increases, N decreases.

If , N0 = initial number or organisms and ,

Nt = number or organisms still present after a time period of t,

Nt = N0 e-kt ………………………(2)
Also:
ln(Nt /N0) = -kt ………………….(3)
or,
ln(N0/Nt) = kt ………………….(4)
 N0/Nt = known as inactivation factor

– Design criteria which indicates the contamination level

of the medium
– Initially present as N0 and need to be brought to a
sterility level of Nt
 a) b) c)
As time of sterilization Decline of ratio Nt/No in Value of –k as its slope.
increases, the number an exponential fashion Complete 100%
of surviving spores as the time proceeds sterilization (i.e Nt = 0) is
decreases Nt = N0 e-kt ……………(2) never possible; or in
-dN/dt = kN………….(1) other words, it takes an
infinite amount of time
to get Nt=0
ln(Nt /N0) = -kt ………….(3)
Value of k in equation (c), obtained in -VE slope is dependent on
type of cells (i.e. species of the cells) and physiological form of the cells.

Typical data of N/N0 vs. sterilization time for spores of Bacillus

stearothermophillus, one of the hardest spores to kill, and vegetative cells
of E. coli
 EXAMPLE
A fermentation medium contains an initial spores
concentration of 8.5x1010. The medium is sterilized
thermally at 120oC, and the pores density was noted with
the progress of time. The data are as follows:

Time 0 5 10 15 20 30
(min)
Spore 8.5x1010 4.23x109 6.2x107 1.8x106 4.5x104 32.5
density
(m-3)

i) Find the thermal death kinetic rate constant in s-1

ii) With the above data, calculate the inactivation factor at 40 min
iii) Calculate the survival factor at 40 min.
Solution:
It is a straight application of equation (2) Nt = N0 e-kt
Prepare the data from table above to draw the plot between ln (Nt/No) and t as
follows:

t(min) Nt Nt/No ln(Nt/No)

0 8.5x1010
5 4.23x109
10 6.2x107
15 1.8x106
20 4.5x104
30 32.5

Draw the graph using data in the first and last column in table above
• From figure above, the slope of the line gives the value of kd in the unit
of min-1
Slope = - kd
= -0.723 min-1
kd = 0.723 min-1 (0.012 s-1 )
ii) Inactivation factor at 40 min,
Nt=No e-0.723 x 40
= 8.5 x 1010 x 2.75 x 10-13
= 0.023
STERILIZATION OF CULTURE MEDIA
Degradation of heat labile compounds
• Different compounds will have different
sterilization periods and temperature
profile
• Temperature effect can be assessed by
Arrhenius equation

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• Assume only 2 organisms, A and B, are
present in the culture which is being
subjected to sterilization.
• Organism A have higher activation
energy EA as compared to that of B
which has activation energy EB
• Plot ln k versus 1/T for A and B (Fig
6.3)
• Slope of the graph = activation energy
of destruction
• Any increase in T, greater effect on A
compared to B (from steeping of plot A
relative to that of B)
• To achieved higher destruction of A ,
the medium can be heated to a higher T
for shorter periods, HTST.
• Use continuous sterilization
• Increase in T will destroy more A
compared to B 31
 BATCH STERILIZATION
• DESIGN ASPECTS
• Using Arrhenius Equation
d(ln k) = E ……… (5)
dT RT2
E = activation energy (in J/mol), a specific constant
for the population
R= universal gas constant (8.314 J/(mol K))
T= temperature (in K)
• Then, integration of equ (5), yields,
k = ko e –E/RT …………………. (6)
ko = Arrhenius constant

32
• Constant ko and E/R can be
calculated by Arrhenius plot draw
between ln k and 1/T (shown in Fig.
6.5)
• Value of k are evaluated by Eq. (3) by
measuring Nt, at different time interval
ln(Nt /N0) = -kt ………………….(3)
• Procedure is repeated at different
other T, and the Arrhenius plot is made
as given in Figure 6.5
• Slope, m = -E/R, intercept at y-axis,
c = ln ko
• T effect on Nt is evaluated and
incorporated in Eq (4) by combining it
with eq (6), get
ln(No/Nt) = ko t e -E/RT……….(7)

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 DEL FACTOR
• Equation (7) act as the design criterion which is called as
‘Del Factor’ (∇)
ln(No/Nt) = ko t e -E/RT……….(7)
• Nabla factor, sterilization criterion
• Defined as – the measure of fractional reduction in living
organisms count over the initial number present
• Produced by a certain heat and time regime
∇ = ln No/Nt …… (8)
Combine eq (7) and (8), give:
∇ = ko t e -E/RT ……….(9)
Rearrangement:
ln t = (E/R) (1/T) + ln (∇ / ko)
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Plot ln (t) versus (1/T)

Degree of sterilization (∇) maybe obtained over a wide range

of t and T regimes

35
 DEL FACTOR
• Practical purpose – a risk factor of 1 in 1000 being
contaminated
• Assume all contaminants under a single category of
spores:
• Bacillus stearothermophilus
That is the final
• The most heat-resistant microbial type microbial count in
• Activation energy, E = 283 kJ/mol the medium after
sterilization should
• Arrhenius constant, ko = 1 x 1036.2 s-1 be 10-3 viable cells

• So, Nt = 10-3
• Del factor, ∇ = ln (No/Nt )
• If unsterile broth contains initially, 1011 viable cells
No = 1011
∇ = ln (No/Nt) = ln (1011 / 10-3 )
= ln (1014 ) = 32.2 36
 DEL FACTOR
• 32.2 is overall del factor
• Normally the destruction of cell takes place at 121oC
(corresponding of steam pressure of 15 psig, which is
equivalent to 0.1 MN/m2 gauge)
• This period known as ‘HOLDING PERIOD’ since the
medium is held at 121oC
• Some cells destroyed during T build-up to 121oC, known
as ‘HEATING PERIOD’
• Some cells destroyed during ‘COOLING PERIOD’ from
121oC to room temperature
• The OVERALL PERIOD is a sum of heating period,
holding period and cooling period
• ∇overall = ∇ heating + ∇ holding + ∇ cooling ….. (10)
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 EXAMPLE PROBLEM
• The thermal death kinetic data of Bacillus
stearothermophilus are as follows at three
different temperatures
T oC 115 120 125
kd, min -1 0.035 0.112 0.347

1) calculate activation energy (E) and Arrhenius

constant (ko) for sterilization
2) find kd at 130oC

38
 solution
Temp (oC) kd, min-1 Temp, K 1/T ln (kd)

115 0.035

120 0.112

125 0.347

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40
 Discussion Example Part 1
• The specific death constants of heating
and cooling during sterilization of a
medium at 121oC are 0.1 min-1 and 0.2
min-1, respectively.
t heating = 20 min
t holding = 30 min
t cooling = 30 min
The decimal reduction time during holding is 2 min.
the initial batch contain 6x1015 organism at 30oC,
find the sterilization performance.

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 Discussion Example Part 2

• In Example Par 1, if the power fails after

15 min of holding time (i.e. after it reaches
121oC), what will happen to the
sterilization if the cooling process is not
affected by it?

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Solution:
• During holding time, the power went off after 15 minutes and so
heating stopped and cooling started.
t holding = 30 – 15 = 15 min
And cooling time increases by 15 min
t cooling = 30 + 15 = 45 min
(No) holding = 8.12 x 1014
(N t) holding = (No) holding e –kdt
= 8.12 x 1014 e –(1.152 x 15)
= 8.12 x 1014 x 3.129 x 10-8 = 2.54 x 107
Hence,
(No) cooling = 2.54 x 107
(Nt) cooling = (No) cooling x e –(0.2(30+15))
= 2.54 x 107 x 1.234 x 10-4 = 3.135 x 103
The number of cells surviving after the sterilisation = 3.13 x 103
Hence the sterilisation is not complete
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