ASTU

Adama Science and Technology University

Department of Chemical Engineering
ChE 4201 Fundamentals of
Biochemical Engineering
Eba A
Course Contents
1. Biotechnology and Biochemical Engineering
2. Enzyme Kinetics
3. Immobilized Enzyme
4. Industrial Applications of Enzymes
5. Cell Kinetics and Fermenter Design
6. Sterilization
7. Agitation and Aeration
8.Downstream Processing
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Objectives & Competences to be Acquired
Upon completion of this chapter, the students
will be able to
 Explain purposes of sterilization in bioprocessing
Explain different techniques for sterilization
 design sterilizer for industry scale sterilization
purpose
 explain kinetics for death of microorganism
during sterilization.
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6. Sterilization
 Sterilization is the process designed to produce a sterile state.
Absolute condition of total destruction or elimination of all living
microorganisms.
 Aseptic condition indicates a controlled process or condition in which
the level of microbial contamination is reduced to the degree that
microorganisms can be excluded from a product during processing.
It describes an "apparently“ sterile state.
 Sterilization of all medium and fermentation equipment should be
done to avoid growth of un needed organisms.
Disinfection refers to the process of eliminating or reducing harmful
microorganisms from inanimate objects or surfaces
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Sterilization Methods
Sterilization can be done by the following methods.
 Thermal Sterilization
 Sterilization By Chemical Agents
 Sterilization by Radiation
 Sterilization By Mechanical Means
 Sterile Filtration
 High Speed Centrifugation

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Sterilization Methods: Thermal sterilization
• Involves the use of either moist or dry heat.
• Moist –heat sterilization is the most widely used and reliable sterilization method.
• Dry – heat sterilization is appropriate for materials that cannot withstand moist –
heat sterilization
In Moist heat sterilizer,
• Microorganism are destroyed by cellular protein coagulation .
• The objects to be sterilized are exposed to saturated steam under 1 atmosphere
pressure at a minimum temperature of 121°C for at least 20-60 minutes.
• Because it does not require as high a temperature, moist – heat sterilization
cause less product and equipment damage compared to dry – heat sterilization
Dry – heat sterilization is appropriate for materials that cannot withstand moist –
heat sterilization (e.g., oily materials and powders) .
• Objects are subjected to a temperature of at least 160 °C for 120 minutes ( if
higher temperatures can be used , less exposure time is required).
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Sterilization Methods-Chemical sterilization
Is used to sterilize surfaces and porous materials ( e.g.,
surgical dressings ) that other sterilization methods may
damage
Residual gas must be allowed to dissipate after sterilization and
before use of the sterile product .
This method is generally used for disinfection( reduction of the risk of
contamination)
1. Phenol and phenolic cpds( phenol, cresol,orthophenylphenol)
2. Alcohols (ethyl, methyl)
3. Halogens(iodine, hypochlorites, chloramines)
4. Detergents. Dyes
5. Quaternary ammonium compounds
6. Gaseous chemosterilizers( ethylene oxide, Formaldehayde)
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Sterilization Methods- Radioactive sterilization
 Is suitable for the industrial sterilization of contents in sealed packages
that cannot be exposed to heat ( e.g. prepackaged surgical
components , some ophthalmic ointments )
 Uv Disinfection
- damage DNA resulting in cell death
- wavelength 265nm is of the highest bactericidal efficiency
- used to reduce microbial population
 X- rays: lethal to most microorganism but not recommended due to
safety concerns.
 Sonic/Ultrasonic Waves: can disrupt and kill cells
used for disruption of cells for the purpose of extracting intracellular
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Sterilization Methods – Sterile filtration)
• Removes but does not destroy microorganisms and clarifies solutions by
eliminating particulate matter.
• For solutions rendered unstable by thermal, chemical, or radiation
sterilization, filtration is the preferred method .
• A depth filter or screen filter may be used .
• Depth filter usually consist of fritted glass or unglazed porcelain (i.e.,
substances that trap particles in channels ).
• Screen (membrane) filters are films measuring 1-200 mm thick made of
cellulose esters, microfilament, polycarbonate , synthetics polymers,
silver, or stainless steel.

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Thermal Death Kinetics
• The thermal death of microorganisms at a particular temperature is given by 1st
order kinetics as below.
• where, Kd is specific death rate.
n is number of viable microorganism

Specific death rate = f( types of species, physiological form of cells)

= 1/min for bacteria spores @ 121 ℃
= 10- 1010 /min for vegetative cell

Integration of the above equation yields which shows the

exponential decay of the cell population.
Temperature dependence of specific death rate is given by Arrhenius equation:
where Ed is activation energy, R is gas constant
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 Design Criterion
 Design criterion for sterilization can be described interms of Del factor 𝛻
Del factor, 𝛻 is give as

Del factor is a measure of the size of the job to be accomplished.

Del factor increases as the final number of cells decreases
 The del factor to reduce the number of cells to zero is infinity: which
means that it is theoretically impossible to ensure the total destruction of
viable cells.
Therefore, The final number of cells needs to be expressed as the fraction
of one, which is equal to the probability of contamination.
Based on the sterilization criterion calculated we can design the
sterilization unit.
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Sterilization Modes: Batch Sterilization
 Sterilization can be done batch wise or continuously.
 In batch sterilization, the entire volume of media is sterilized at once
through the use of different sterilization methods. Batch sterilization is done
using the following major methods
1. Direct steam sparging
2. By electrical heaters
3. By circulating constant pressure condensing steam through heating coil
Total sterilization cycles = Heating + holding + Cooling
Total Del factor = 𝛻heating+ 𝛻 holding + 𝛻 cooling
Del factor for heating and cooling are determined by the methods used for
heating and cooling
Del factor for holding is determined by the length of the controlled holding
period
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Design Procedure for Estimation of Holding Time
1. Calculate the total sterilization criterion, 𝛻 total.
2. Measure the temperature versus time profile during the heating, holding, and
cooling cycles of sterilization. If experimental measurements are not practical,
theoretical equations for heating and cooling can be employed, which are of
linear, exponential, or hyperbolic form depending on the mode of heating and
cooling.
a. For batch heating by direct steam sparging into the medium, the hyperbolic
𝐻𝑚𝑠𝑡
form is used: T= 𝑇𝑜 +
𝑐 𝑀+𝑚𝑠𝑡
b. For batch heating with a constant rate of heat flow such as electrical heating, the
𝑞𝑇𝑡
linear form is used: 𝑇 = 𝑇𝑜 + 𝑐𝑀
c. For batch heating with a isothermal heat source such as steam circulation through
heating coil, the exponential form is used:
𝑈𝐴𝑡
𝑇 = 𝑇ℎ + 𝑇𝑜 − 𝑇ℎ exp −
𝑐𝑀 13
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Design Procedure for Estimation of Holding Time
3. For batch cooling using a continuous non-isothermal heat sink such as
passing cooling water through cooling coil, the exponential form is
𝑈𝐴 𝑚𝑐𝑡
used: 𝑇 = 𝑇𝑐𝑜 + 𝑇𝑜 − 𝑇𝑐𝑜 exp{− 1 − exp − }
𝑚𝑐𝐶 𝑀

4. Plot the values of Kd as a function of time.

5. Integrate the areas under Kd Vs time curve for heating and cooling
periods to estimate 𝛻 heating and 𝛻 cooling respectively.

thold= 𝛻total/ Kd= [ 𝛻 heating +𝛻 holding +𝛻 cooling ]/ Kd

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Continuous Sterilization
Sterilization can be carried out in a continuous mode rather than in batches.
This offers several advantages over batch sterilization.
I. It simplifies production planning, thus allowing maximum plant
utilization and minimum delays.
II. It provides reproducible conditions.
III. It can be operated at a high temperature (140°C instead of 121°C in
batch sterilization); therefore, the sterilization time can be shortened
(holding time of 1 to 2 minutes).
IV. It requires less steam by recovering heat from the sterilized medium. As a
result, it also requires less cooling water.
V. 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.
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Continuous Sterilization- Heating Section
Heating can be done by direct steam
injection or indirect heating(PHE and
STHE)
Direct heating heats the media to the
peak sterilization temperature quickly
PHE is more effective than STHE due
to its high surface area.  For heating using a
countercurrent heat source
Temperature change w.r.t residence
of equal flowrate and heat
time as the medium passes through an capacity
isothermal heat source is ∆𝑇𝑈𝐴𝜏ℎ𝑒𝑎𝑡
𝑈𝐴𝜏ℎ𝑒𝑎𝑡 𝑇𝑐2 = 𝑇𝑐1 −
𝑇𝑐2 = 𝑇ℎ − 𝑇ℎ − 𝑇𝑐1 exp(− ) 𝑐𝑊
𝑐𝑊
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Continous Sterilization- 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:
𝜏ℎ𝑜𝑙𝑑 = 𝐿/𝑈
Del factor can be estimated as
Where no is the number of cells at the beginning of the holding sections
For ideal plug flow, residence time will be the same for all sections.
Thus , uniform degree of sterilization is assumed to be achieved.

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Continuous Sterilization- Holding Section
How ever, the flow is not ideal plug flow due to the following factors.
 slippage due to viscous nature of the fluid
 the friction of the pipe wall
 Turbulent eddies of the flowing fluid
Velocity profile is maximum at the centerline and minimum at the vicinity of
the pipe wall.
For laminar flow: Average velocity/Max velocity: u/umax = 0.5
For turbulent flow: u/umax = 0.5 – 0.75
For Re>1000,000 : u/umax = 0.87
As a result, if we use the mean velocity um in calculating the required
residence time for sterilization, some portion of the medium will be under
sterilized which may cause a serious contamination problem. See(Example
8.2)
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Continuous Sterilization- 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.
• The temperature versus residence time relationship for cooling using
𝑈𝐴𝜏𝑐𝑜𝑜𝑙
isothermal heat sink is 𝑇ℎ2 = 𝑇𝑐 − 𝑇𝑐 − 𝑇ℎ1 exp(− )
𝑐𝑊
• For a cooling using a countercurrent heat sink of equal equal flow
rate and capacity.
𝑇ℎ2 = 𝑇ℎ1 − ∆𝑇𝑈𝐴𝜏𝑐𝑜𝑜𝑙/𝑐𝑊
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Air Sterilization
 Aerobic fermentation requires air to proceed. Typical air flowrates is
0.5 -1.0vvm( air volume per liquid volume per minute).
• Sterilization of air by heat is economically impractical and ineffective
due to the low heat transfer efficiency of air compared with those of
liquid.
• The most effective techniques are:
Filtration using fibrous(membrane filters) eg. Cotton plug (closure of
tubes/flasks)
 glass fibers
 With fibrous filters, airborne particles are collected by the
mechanisms of impaction, interception and diffusion
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Air Sterilization Cont’d
 Impaction: When air stream containing particles flows around
cylindrical collector, the particle will follow the streamlines until they
diverge at the collector.
 Interception: This is considered where the particle has size , but no
mass, so they can follow the streamlines of the air around the
collection.
 Diffusion: Particles smaller than about 1micron in diameter exhibit a
Brownian motion which is sufficiently intense to produce diffusion.
Collection efficiency increases as the particle size decreases.
 Total collection efficiency of a fibrous filter is obtained from the
combined effect of interception, impaction and diffusion.

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THE END !

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