INDUSTRIAL BIOTECHNOLOGY

Lecture 16: Life cycle of the microbial cell, Microbial growth kinetics, product formation and
substrate degradation (continued)

PROF. DEBABRATA DAS

DEPARTMENT OF BIOTECHNOLOGY
IIT KHARAGPUR

1
Comparison between cascade chemostat and plug flow
reactor
 It can also be shown theoretically that as the number of stages in a
chemostat (CSTR) cascade increase, the conversion characteristics of the
entire system approach
0 those of an ideal plug flow or mixed batch reactor

Bioprocess Engineering Principles by Pauline M. Doran

Prof. Debabrata Das
Department of Biotechnology
Comparison between cascade chemostat and plug
flow reactor
 Smooth dashed curve represents the progressive decrease in substrate
concentration with time spent in plug flow reactor

 To eliminate product inhibition problem, plug flow reactor is to be selected

but operation and control are very difficult in case of plug flow reactor

 For most fermentation process, chemostat offer significant theoretical

advantages over PFR and batch reactor

Prof. Debabrata Das

Department of Biotechnology
CSTR with cell mass recycling

Prof. Debabrata Das

Department of Biotechnology
 Chemostat with recycle
 To keep the cell concentration higher than the normal steady-state level in
a chemostat, cells in the effluent can be recycled back to the reactor

 Cell recycle increases the rate of conversion (or productivity)

 Increase stability by dampening perturbations of input stream properties

 Cell mass balance around the chemostat

=0
Input + cell generation = output + cell death + accumulation

Prof. Debabrata Das

Department of Biotechnology
 Centrifuge
Micro-filter
Settling tank

Bioprocess Engineering
Basic Concepts Second
Edition. Shuler and
Kargi

Prof. Debabrata Das

Department of Biotechnology
 Prof. Debabrata Das
Department of Biotechnology
Fresh Recycled Out stream
feed feed from reactor consumption

Prof. Debabrata Das

Department of Biotechnology
 Prof. Debabrata Das
Department of Biotechnology
 Prof. Debabrata Das
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 Prof. Debabrata Das
Department of Biotechnology
Chemostat with recycle
With recycle
1

Without recycle

Bioprocess Engineering Basic Concepts Second Edition. Shuler and Kargi

Prof. Debabrata Das

Department of Biotechnology
 Multistage chemostat in series
 For secondary metabolite production, the growth and product-formation steps
need to be separated, since optimal conditions (temperature, pH and limiting
nutrients) for each step are different. As a result multistage chemostat is
employed

 Assumption

 Steady state operation

 No substrate addition in the consecutive stages

 Balanced growth in all the stages

Prof. Debabrata Das
Department of Biotechnology
 =0 =0

Bioprocess Engineering Principles by Pauline M. Doran

Prof. Debabrata Das

Department of Biotechnology
 Prof. Debabrata Das
Department of Biotechnology
 Prof. Debabrata Das
Department of Biotechnology
 For negligible
=0 endogeneous
metabolism, death→0

Prof. Debabrata Das

Department of Biotechnology
 Prof. Debabrata Das
Department of Biotechnology
Hydraulic retention time (HRT) =

Solid retention time (SRT) = mean cell residence time

In case of CSTR, HRT = SRT

In case of CSTR with cell mass recycling, SRT > HRT

Prof. Debabrata Das
Department of Biotechnology
 Prof. Debabrata Das
Department of Biotechnology
 Prof. Debabrata Das
Department of Biotechnology
 Prof. Debabrata Das
Department of Biotechnology
 Prof. Debabrata Das
Department of Biotechnology
 Prof. Debabrata Das
Department of Biotechnology
 Prof. Debabrata Das
Department of Biotechnology
 S (mg/L) X (mg/L)
0.3 45 326
0.25 41 328
0.20 16 340
0.12 8 342
0.08 3.8 344

Prof. Debabrata Das

Department of Biotechnology
 Prof. Debabrata Das
Department of Biotechnology
0.0222 3.33
0.0244 4.00
0.0625 5.00
0.1250 8.33
0.2632 12.50

Prof. Debabrata Das

Department of Biotechnology
 Prof. Debabrata Das
Department of Biotechnology
 Prof. Debabrata Das
Department of Biotechnology
 S (mg/L) X (mg/L)

0.3 3.33 45 326 0.498 2.008

0.25 4.00 41 328 0.4977 2.009
0.20 5.00 16 340 0.4970 2.012
0.12 8.33 8 342 0.4942 2.023
0.08 12.50 3.8 344 0.4941 2.024

Prof. Debabrata Das

Department of Biotechnology
 Prof. Debabrata Das
Department of Biotechnology
Problem:

Consider an organism which follows the Monod equation where μmax =

0.5 h-1 and Ks = 2 g/L. In a continuous perfectly mixed vessel at steady
state with no cell death, if So = 50 g/L and Yx/s = 1, what dilution rate D
will give the maximum total rate of cell production? For the same value
of D using tanks of the same size in series, how many vessels will be
required to reduce the substrate concentration to 1 g/L?

Prof. Debabrata Das

Department of Biotechnology
 Prof. Debabrata Das
Department of Biotechnology
 Prof. Debabrata Das
Department of Biotechnology
 Prof. Debabrata Das
Department of Biotechnology 36
INDUSTRIAL BIOTECHNOLOGY
Lecture 17: Overview of the fermenter

PROF. DEBABRATA DAS

DEPARTMENT OF BIOTECHNOLOGY
IIT KHARAGPUR

1
Schematic diagram of the Bioreacter

Singh J. Kaushik N. and Biswas S. Bioreactor- technology and design analysis. The scitech J. 2014. v1(06)
Prof. Debabrata Das
Department of Biotechnology
 Laboratory fermenter
Schamatic diagram of stirred tank photobioreactor (Model:
3.7 KLF 2000, BioEngineering AG, Switzerland). Photo
courtesy (BioEngineering AG, Switzerland). 1) Exhaust
airfilter, (B, Exhaust air); 2) Safety valve; 3) Safety jacket; 4)
Glass cylinder; 5) Cooling finger (C, Cooling water inlet; D,
Cooling water outlet); 6) Turbo stirrer; 7) Stirring shaft; 8)
Heating finger; 9) Temperature probe Pt 100; 10)
Hypodermic needle; 11) Non-return valve; 12) Aeration tube;
13) Air inlet filter (A, Air inlet); 14) Harvest valve; 15)
Bearing; 16) Leakage cup; 17) Motor.

Prof. Debabrata Das

Department of Biotechnology
 Stirred tank
Photobioreactor
(Model: 3.7 KLF 2000,
BioEngineering AG,
Switzerland)

Prof. Debabrata Das

Department of Biotechnology
Laboratory fermenter

Prof. Debabrata Das

Department of Biotechnology
Industrial fermenter

[Link]

Prof. Debabrata Das

Department of Biotechnology
Accessories of the fermenter

Aeration Loop
The primary purpose of aeration is to provide microorganisms in
submerged culture with sufficient oxygen for metabolic
requirements.

(i) Compressor
It is a mechanical device that increases the pressure of a gas by
reducing its volume and forces more and more gas in the storage
tank.
Prof. Debabrata Das
Department of Biotechnology
(ii) Rotameter
A device that measure the flow rate of fluid in a closed tube
by allowing the cross-sectional area the fluid travels through
to vary causing a measurable effect.

[Link]

Prof. Debabrata Das

Department of Biotechnology
(iii) Air filter
Air out
An air filter is a device composed of fibrous materials which removes solid particulars such
as dust pollen, mould and bacteria from air.
Fibrous materials
(iv) Non-return valve
Also called one-way valve normally allows fluid to flow through it in only one direction. The
Air in
purpose of the check valves is to prevent the accidental reversal flow of liquid or gas in a
pipe due to breakdown back to the pump.

(v) Mechanical Seal

The seal consists of two pars, the stationary part in the
bearing and the other rotating on the shaft. The two
components are pressed together by springs. Sterilized
antifoam oils are used to lubricate and cool seals during
operations. [Link]
Prof. Debabrata Das
[Link]

Department of Biotechnology
(vi) Fermenter vessel
For small scale fermentation glass vessels are used. The volume of the
one in lab is 3.4 L. Glass vessels are usually smooth which makes it
nontoxic & corrosion proof. It is easy to examine the interior of the
vessel. These vessels require autoclaved sterilization.

(vii) Feed ports

They are silicon tubes connected to the nutrient reservoir. They are used
to add nutrients and other important substituent in the fermenter
vessel.

Prof. Debabrata Das

Department of Biotechnology
 (viii) Baffles
Metal strip attached radically to the wall. They are used to prevent vortex and to
improve aeration capacity. Baffles maintain a gap between them and the vessel
wall to enable scouring action thus minimising microbial growth on the walls of
fermenter.

[Link]

(ix) Impeller
They are used for agitation which is required to
ensure that a uniform suspension of microbial cells
is achieved in a homogeneous nutrient medium. Rushton impeller
[Link]
Prof. Debabrata Das
Department of Biotechnology
(x) Sparger
It is used for aeration. The purpose of aeration is to provide sufficient oxygen to the
microorganism for metabolic requirements sparger introduces air into liquid in the
vessel. Sparger can be of three types: porous, orifice, nozzle.

[Link]

(xi) Outlet
Reflux cooler: The air flowing out of the fermenter has the same temperature as
during cultivation and is also correspondingly saturated. The moisture is condensed
out with reflux cooler and then is returned to the fermenter.
Outlet valve: Regulates the release of air or liquid from the fermenter.

Prof. Debabrata Das

Department of Biotechnology
Exhaust Air Filter:
Used to filter the air that is going out of the fermenter in order to prevent
the environment from being contaminated by any biohazards that may be
produced. During power cut, the inlets is closed, and then exhaust air filter
supplies filtered air required for survival of cells.

(xii) Safety valve:

These valves ensure that the pressure never exceeds the safe upper limit of
the specified valve.

[Link]

Prof. Debabrata Das

Department of Biotechnology
Temperature Loop

Heater: Heat is generally provided to the fermenter by internal coils

which gets heated up and directly heats the medium. Thermostatically
controlled bath can also be used.

Cold finger: Closed coil or pipe in which coolant liquid (cold water
etc.) can enter and leave. It is used to generate generalized cooling.

Prof. Debabrata Das

Department of Biotechnology
pH probe:

To monitor the pH of
the culture broth
during the course of
growth of microbes
and reaction in the
vessel.

Biochemical Engineering by Aiba, Humphrey and Millis

Prof. Debabrata Das

Department of Biotechnology
 Prof. Debabrata Das
Department of Biotechnology
 Instrumentation and control
Temperature probe: used to measure the temperature of the culture broth in
the (Pt 100) fermenter vessel.
O2 sensor: Monitor dissolved oxygen level in the system.
Motor: Provide energy for the impeller to stir the culture motors can be bottom
driven or top driven.
Foam Control: A probe (foam sensing and control unit) is inserted through the
top plate. It is set at a defined level above the broth surface, when the foam rise
and touches the probe surface/tip, a current is passed through the circuit which
actuates the pump and antifoam is released within seconds.

Prof. Debabrata Das

Department of Biotechnology
 Prof. Debabrata Das
Department of Biotechnology 18
 INDUSTRIAL BIOTECHNOLOGY
Lecture 18: Flow diagram and pumps and valves used in Fermentation industries

PROF. DEBABRATA DAS

DEPARTMENT OF BIOTECHNOLOGY
IIT KHARAGPUR

1
 Flow diagram
 The most effective way of communicating information about a process is
through the use of flow diagrams

Flow diagram

Piping &
Block flow Process flow
instrumentation
diagram diagram
diagram

Prof. Debabrata Das

Department of Biotechnology
Block flow diagram

A block flow diagram (BFD) is a drawing of a chemical /

biochemical processes used to simplify and understand the
basic structure of a system. A BFD is the simplest form of
the flow diagrams used in industry. Blocks in a BFD can
represent anything from a single piece of equipment to an
entire plant.

Prof. Debabrata Das

Department of Biotechnology
Block flow diagram of Citric acid purification process

Prof. Debabrata Das

Department of Biotechnology
Process flow diagram
The Process Flow diagram provides a visual representation of the steps in a
process. Flow diagrams are also referred to as Process Mapping. It has the
following benefits:
 Gives everyone a clear understanding of the process
 Helps to identify non-value-added operations
 Facilitates teamwork and communication
 Keeps everyone on the same page

Prof. Debabrata Das

Department of Biotechnology
 Process flow diagram (PFD)

 A process flow diagram (PFD) is a diagram commonly used in process

engineering to indicate the general flow of plant processes and equipment

 The PFD displays the relationship between major equipment of a plant

facility and does not show minor details such as piping details and
designations

 Another commonly used term for a PFD is a flow-sheet

Prof. Debabrata Das

Department of Biotechnology
Process Unit Symbols for PFD

Prof. Debabrata Das

Department of Biotechnology
Process Unit Symbols for PFD

Prof. Debabrata Das

Department of Biotechnology
Process Unit Symbols for PFD

Prof. Debabrata Das

Department of Biotechnology
Process Unit Symbols for PFD

Prof. Debabrata Das

Department of Biotechnology
Process Unit Symbols for PFD

Prof. Debabrata Das

Department of Biotechnology
Process Unit Symbols for PFD

Prof. Debabrata Das

Department of Biotechnology
Valves Symbols for PFD

Prof. Debabrata Das

Department of Biotechnology
Valves Symbols for PFD

Prof. Debabrata Das

Department of Biotechnology
Ethanol fermentation from corn starch

[Link]

Prof. Debabrata Das

Department of Biotechnology
Piping and Instrumentation diagram (P & ID)

 A piping and Instrumentation diagram/drawing (P&ID) is a detailed

diagram in the process industry which shows the piping and vessels in the
process flow, together with the instrumentation and control devices.

 The primary schematic drawing used for laying out a process

control installation

Prof. Debabrata Das

Department of Biotechnology
 Utilities of fermentation industries
 Pumps
 A pump is a device that moves fluids (liquids or gases), or
sometimes slurries, by mechanical action
 Used for-
• Domestic, commercial, industrial and agriculture service
• Municipal water and wastewater service

 The pumps can be classified in to two major types according to

principle by which energy is added to the fluid
Prof. Debabrata Das
Department of Biotechnology
 Classification of pumps

Prof. Debabrata Das

Department of Biotechnology
[Link]
 Positive displacement pump
 Positive Displacement pumps apply pressure directly to the liquid
by a reciprocating piston, or by rotating members
 Uses
• can handle shear sensitive liquid.
• Use for high pressure application
• Use for variable viscosity applications
 Types
• Reciprocating
• Rotary
Prof. Debabrata Das
Department of Biotechnology
 Reciprocating pump
 In Reciprocating pumps, the chamber is a stationary cylinder
that contains a piston or plunger
 Types
• Diaphragm
• Piston

Piston pump
Diaphragm pump [Link]
[Link]
Prof. Debabrata Das
Department of Biotechnology
 Rotary pump
 In Rotary pumps, the chamber moves from inlet to discharge
and back to the inlet.
 A wide variety of rotary pumps are available like gear pumps,
lobe pumps, peristaltic pump, screw pumps, cam pumps, vane
pumps.
 Relatively constant output
 Most popular –peristaltic pump(mostly used in biological
process), gear pump(highly viscous fluid)

Prof. Debabrata Das

Department of Biotechnology
 Peristaltic pump
Gear pump

[Link]
[Link]
Prof. Debabrata Das
Department of Biotechnology
 Dynamic pump

 Dynamic pumps are a type of velocity pump in which kinetic

energy is added to the fluid by increasing the flow velocity.

 Types-
• Centrifugal pump
• Propeller
• Turbine

Prof. Debabrata Das

Department of Biotechnology
  Centrifugal pump
 This pumps generate high rotational velocities, then convert the resulting
kinetic energy of the liquid to pressure energy
 Uses-
• Centrifugal pumps are generally use where high flow rates and moderate
head increases are required
• Can handle fluids containing suspended solids

Centrifugal pump
[Link]
[Link]
Prof. Debabrata Das
Department of Biotechnology
 Valves
 A valve is a device that regulates, directs or controls the flow of a fluid
(gases, liquids, fluidized solids, or slurries) by opening, closing, or partially
obstructing various passageways.
 Valves can be operated by
• Manually
• Pneumatic pressure
• Motor
 Basic valve components-
• Body
• Bonnet
• Trim
• Packing [Link]
• Actuator Prof. Debabrata Das
Department of Biotechnology
 Classification of valve
 According to the motion of valve stem, it can be classified into
two types

[Link]

Prof. Debabrata Das

Department of Biotechnology
 Globe valve
 Globe valves restrict the flow of fluid by altering the distance
between a movable plug and a stationary seat (in some cases,
a pair of plugs and matching seats )

[Link]

Prof. Debabrata Das

Department of Biotechnology
 Diaphragm valve

 Diaphragm valves use a flexible sheet pressed close to the edge

of a solid dam to narrow the flow path for fluid.
 These valves are well suited for flows containing solid
particulate matter such as slurries.

[Link]

Prof. Debabrata Das

Department of Biotechnology
[Link]
 Utilities for reactors
 Gate valve
 Gate valves work by inserting a dam (gate) into the path of the flow to
restrict it, in a manner similar to the action of a sliding door.

 Gate valves are more often used for on/off control than for throttling

[Link]

Prof. Debabrata Das

Department of Biotechnology
 Utilities for reactors
 Ball valve
 A spherical ball with a passageway cut through the centre
rotates to allow fluid more or less access to the passageway
 It can be used for sample collection

[Link]

Prof. Debabrata Das

Department of Biotechnology
 Butterfly valve
 The “butterfly” element is a disk that rotates perpendicular to the path of
fluid flow
 When the disc is parallel to the axis of flow, the disk presents minimal
obstruction; when perpendicular to the axis, the disk completely blocks any
flow
 Used in large pipeline

open
closed

[Link]

Prof. Debabrata Das

Department of Biotechnology
 Disk valve
 Often referred to as eccentric disk valves, or as high-
performance butterfly valves
 The disk’s centre is offset from the shaft centreline, causing it to
approach the seat with a “cam” action that results in high
seating pressure

[Link]

Prof. Debabrata Das

Department of Biotechnology
 Other types
 Needle valve
 A needle valve is a type of valve having a small port and
a threaded, needle-shaped plunger.
 It allows precise regulation of flow, although it is generally
only capable of relatively low flow rates
 Used in precious application

[Link]

Prof. Debabrata Das

Department of Biotechnology
 Safety valve
 A safety valve opens slowly as the pressure increases about the
set point and only opens as necessary

[Link]

Prof. Debabrata Das

Department of Biotechnology
Solenoid valve

• A solenoid valve is an electromechanically operated valve.

• In the case of a two-port valve the flow is switched on or off;
• In the case of a three-port valve, the outflow is switched between the
two outlet ports.
• Multiple solenoid valves can be placed together on a manifold.

[Link]

Prof. Debabrata Das

Department of Biotechnology
Pneumatic Valve
The study of pneumatics deals with system
operation with air or gaseous medium to impart
power or to control power.
The term pneumatics is derived from the
Greek word pneuma, meaning wind or breath.
 Pneumatic power is the power that is
transmitted by pressurized/compressed air.

[Link]

Prof. Debabrata Das

Department of Biotechnology
 References
1. [Link]

2. [Link]

3. [Link]

Prof. Debabrata Das

Department of Biotechnology
 Prof. Debabrata Das
Department of Biotechnology 38
 INDUSTRIAL BIOTECHNOLOGY
Lecture 19: Upstream processing: Air sterilizer
PROF. DEBABRATA DAS
DEPARTMENT OF BIOTECHNOLOGY
IIT KHARAGPUR

1
 Air sterilization

 For an effective aerobic fermentation, the air should be completely sterile, and free
from all micro-organisms and suspended particles.

 There is a wide variation in the quantity of suspended particles and microbes in the
atmospheric outdoor air.

 The microorganisms may range from 10-2,000/m3 while the suspended particles may be 20-
100,00/ m3.

 Among the microorganisms present in the air, the fungal spores (50%) and Gram-negative
bacteria (40%) dominate.

Prof. Debabrata Das

Department of Biotechnology
 Airborne microbes

 These mainly consist of species of bacteria, bacterial spores, yeast, fungi and
viruses.

 The size of these organisms varies from 0.5 μm to several hundred μm.

 The airborne particles which have to be destroyed or collected during air

sterilization are about the size of small bacteria, namely 0.5 to 1 μm.

Prof. Debabrata Das

Department of Biotechnology
 Methods of sterilization
 Air can be sterilized by many methods namely-

(i) Filtration
(ii) Heat
(iv) U.V. light
(iv) Chemical agents
Among these, heat and filtration are most commonly used.

Prof. Debabrata Das

Department of Biotechnology
 Filtration
 Two types of filters are used:
 Depth filters:
A Depth Filter is a filter consisting of either multiple layers or a single layer of a
medium having depth, which captures contaminants within its structure, as
opposed to on the surface.

 Membrane Filters:
A Membrane Filter typically traps contaminants larger than the pore size on the
addressed surface of the membrane.

Prof. Debabrata Das

Department of Biotechnology
 Depth filters:
 When the air is passed through a glass wool containing depth filters the
particles are trapped and removed. This filtration technique primarily
involves physical effects such as inertia, blocking, gravity, electrostatic
attraction and diffusion.

[Link]

Prof. Debabrata Das

Department of Biotechnology
 Principles of filter separation:

 Inertial impaction

 Diffusion

Interception

 Electrostatic attraction

 Gravitation
Prof. Debabrata Das
Department of Biotechnology
 Principles of filter separation:
 Assumptions are:
 Single cylindrical fibers are placed perpendicularly to the aerosol flow in
an infinite space, and that the air flow around the cylinder is laminar with
no vortices.

 The following analyses are 2-dimensional.

Prof. Debabrata Das

Department of Biotechnology
 Inertial impaction:
 Suspended particles in a air stream have momentum

 The air in which the particles are suspended will flow through the filter by the
route of least resistance

 However, the particles, because of their momentum, tend to travel in straight

lines

[Link]
Prof. Debabrata Das
Department of Biotechnology
 Prof. Debabrata Das
Department of Biotechnology
 Prof. Debabrata Das
Department of Biotechnology
 Interception:
 The fibers comprising a filter are interwoven to define openings of various
sizes

 Particles which are larger than the filter pores are removed by direct
interception

 However, a significant number of particles which are smaller than the

filter pores are also retained by interception

Prof. Debabrata Das

Department of Biotechnology
 Interception:
 This may occur by several mechanisms - more than one particle may arrive at a
pore simultaneously, an irregularly shaped particle may bridge a pore, once a
particle has been trapped by a mechanism other than interception the pore may
be partially occluded enabling the entrapment of smaller particles

[Link]

Prof. Debabrata Das

Department of Biotechnology
 Prof. Debabrata Das
Department of Biotechnology
 Diffusion:
 Extremely small particles suspended in the air are subject to Brownian
motion which is random movement due to collisions with fluid molecules

 Thus, such small particles tend to deviate from the fluid flow pattern and may
become impacted upon the filter fibers

[Link]
Prof. Debabrata Das
Department of Biotechnology
 Prof. Debabrata Das
Department of Biotechnology
 Electrostatic attraction:
 Charged particles may be attracted by opposite charges on the surface of the
filtration medium

 Microorganisms have different charges on their surface, so it can be neglected

Gravitation:
 As the mass of microorganisms is very less, this can be neglected
Prof. Debabrata Das
Department of Biotechnology
 Prof. Debabrata Das
Department of Biotechnology
 Selection criteria of air-filter

 The criteria involved in selecting a fermentation air filter system for inlet or
exhaust gas filtration are :
 Filter retention efficiency,
 Economy of operation,
 Ease of filter use, and
 Service provided by the manufacturer.

Prof. Debabrata Das

Department of Biotechnology
 Selection criteria of air-filter

 The most important selection criteria are filter efficiency and reliability of
organism retention. In this regard, fixed submicron pore size membrane
filters provide the highest level of filtration efficiency.

 Use of a hydrophobic filtering material minimizes or eliminates concerns of

filter wetting due to air moisture content.

Prof. Debabrata Das

Department of Biotechnology
 Theory of depth filtration
 If it is assumed that if a particle touches a fiber it remains attached to it, and
that there is a uniform concentration of particles at any given depth in the
filter, then each layer of a unit thickness of the filter should reduce the
population entering it by the same proportion; which may be expressed
mathematically as:

dN/dx = -kN
where
N is the concentration of particles in the air at a depth x in the filter
k is a constant.

Prof. Debabrata Das

Department of Biotechnology
 Theory of depth filtration
On integrating above equation over the length of the filter it becomes:
N / No= e-kx.
where
No is the number of particles entering the filter
N is the number of particles leaving the filter.
On taking exponential logarithms:
In (N / No) = -kx
This equation is termed the log penetration relationship.
Consideration of equation indicates that a plot of the logarithm of (N /No)
against x, filter length, will yield a straight line of slope k.
Prof. Debabrata Das
Department of Biotechnology
 Theory of depth filtration
The efficiency of the filter is given by the ratio of the number of particles
removed to the original number present, thus:
E = (No - N)/No
where
E is the efficiency of the filter.
But
(No - N)/No = 1 - (N / No)
Substituting: N/No = e-kx
Thus:
(No - N)/No = 1- e-kx and E = 1 - e-kx.
Prof. Debabrata Das
Department of Biotechnology
 Theory of depth filtration
The log penetration relationship can be used in filter design, by using the
concept X90, the depth of filter required to remove 90% of the total number of
particles entering the filter, thus:
If No is 10 and x is X90 then N would be 1:
In (1/10) = -k X90
2.303 log10 (1/10) = - k X90
2.303(-1) = -k X90

Therefore
X90 = 2.303/k
Prof. Debabrata Das
Department of Biotechnology
 Theory of depth filtration
The value of k is affected by the nature of the filter material and by the linear
velocity of the air passing through the filter.
K increases to an optimum with increasing air velocity, after which any further
increase in air velocity results in a decrease in k.

Prof. Debabrata Das

Department of Biotechnology
 Theory of depth filtration
 The increase in k with increasing air velocity is probably due to increased
impaction, illustrating the important contribution this mechanism makes to
the removal of organisms. The decrease in k values at high air velocities is
probably due to disruption of the filter, allowing channels to develop and
fibers to vibrate, resulting in the release of previously captured organisms

.
Prof. Debabrata Das
Department of Biotechnology
Problem:
It is required to provide a 20 m3 fermenter with air at a rate of 10 m3 min-1 for a
fermentation lasting 100 hours. From an investigation of the filter material to be
used, the optimum linear air velocity was shown to be 0.15 m sec-1, at which the
value of k was 1.535 cm-1. The dimensions of the filter may be calculated as
follows:

The log penetration relationship states that:

In (N / No) = - k x.
The air in the fermentation plant contained approximately 200 microorganisms
m-3.

Prof. Debabrata Das

Department of Biotechnology
Therefore,
No = total amount of air provided X 200
No = 10 X 60 X 100 X 200
= 12 X 106 organisms.
The acceptable degree of contamination is one in a thousand,
Therefore
N = 10-3,
ln(10-3/(12 X 106)) = - k x
In( 8.33 X 10-11 )= - k x,
In (8.33 X 10-11 )= -1.535 x,
x = (-23.21)/(- 1.535) = 15.12 cm.
Prof. Debabrata Das
Department of Biotechnology
Therefore, the filter to be used should be 15.12 cm long.

The cross-sectional area of the filter is given by the volumetric air flow rate
divided by the linear air velocity:
πr2= 10/(0.15 X 60)
Where r is the radius of the filter
r = 0.59 m.
Thus the filter to be employed should be 15.12 cm long and 0.59 m radius.

Prof. Debabrata Das

Department of Biotechnology
 Membrane filters:
 These are fixed pore filters (which have an absolute rating) are very widely
used in the fermentation industry and several manufacturers produce filtration
systems for air sterilization.

 The pre-filter traps large particles such as dust, oil and carbon (from the
compressor) and pipe scale and rust (from the pipework). The use of a
coalescing (combined, united) pre-filter also ensures the removal of water
from the air; entrained water is coalesced in the filter (air flow being from the
inside of the filter to the outside) and is discharged via an automatic drain

Prof. Debabrata Das

Department of Biotechnology
 Membrane filters:
 These filters are made from a variety of polymeric materials such as cellulose
nitrate, cellulose diacetate, polycarbonate and polyester.

 These membranes have a pore diameter ranging from 0.015 μm to 12 μm.

These filters are sterilized by autoclaving.

 Membrane filters are made in two ways, the capillary pore membranes have
pores produced by radiation while the labyrinthine pore membranes are
produced by forced evaporation of solvents from cellulose esters.

Prof. Debabrata Das

Department of Biotechnology
 Prof. Debabrata Das
Department of Biotechnology 32
 INDUSTRIAL BIOTECHNOLOGY
Lecture 20: Upstream processing: medium sterilization

PROF. DEBABRATA DAS

DEPARTMENT OF BIOTECHNOLOGY
IIT KHARAGPUR

1
 Sterilization:
 Destruction or removal of all viable organisms from an object or from a
particular environment.

 The various methods of sterilization are:

1. Physical Method 2. Chemical Method

a. Thermal (Heat) methods a. Alcohols
1. Dry heat b. Oxidizing agents
2. Moist heat c. Salts
b. Radiation method d. Surface active agents
c. Filtration method e. Disinfectants
Prof. Debabrata Das
Department of Biotechnology
 Thermal (Heat) methods :

 Dry heat:
It employs higher temperatures in the range of 160-180˚C and requires
exposures time up to 2 h, depending upon the temperature employed.

 Moist heat:
Moist heat sterilization involves the use of steam in the range of 121-
140˚C. Steam under pressure is used to generate high temperature needed for
sterilization.

Prof. Debabrata Das

Department of Biotechnology
 Radiation method:
 Sterilization can be achieved using electromagnetic radiation such
as electron beams, X-rays, gamma rays, or irradiation by subatomic
particles. Electromagnetic or particulate radiation can be energetic enough
to ionize atoms or molecules (ionizing radiation), or less energetic (non-
ionizing radiation).

 Non-ionizing radiation sterilization

Ultraviolet light irradiation (UV, from a germicidal lamp) is useful for
sterilization of surfaces and some transparent objects. UV irradiation is
routinely used to sterilize the interiors of biological safety cabinets
Prof. Debabrata Das
Department of Biotechnology
 Radiation method:
 Ionizing radiation sterilization
Gamma radiation is very penetrating, and is commonly used for
sterilization of disposable medical equipment, such as syringes, needles
and food. It is emitted by a radioisotopes.

Prof. Debabrata Das

Department of Biotechnology
 Filtration method:
 Filtration process does not destroy but removes the microorganisms. It is
used for both the clarification and sterilization of liquids and gases as it is
capable of preventing the passage of both viable and non viable particles.

 Sterilize solutions that may be damaged or denatured by high temperatures or

chemical agents.

 The major mechanisms of filtration are sieving, adsorption and trapping

within the matrix of the filter material.

 The pore size for filtering bacteria, yeasts, and fungi is in the range of 0.22-
0.45 μm
Prof. Debabrata Das
Department of Biotechnology
Heat sterilization in fermentation industries

 Thermal Death Time (TDT) is the shortest time required to kill all micro-
organisms in a sample at a specific temperature and under defined conditions

 Decimal reduction time (D-value) is the time required to kill 90% of the
microorganisms in a sample at a specific temperature

Prof. Debabrata Das

Department of Biotechnology
 Kinetics of thermal death of microorganisms

The thermal death kinetics may be represented by the following equation:

-dN/dt = kd N (1)

Where,
– N, is the number of viable organisms present,
– t, is the time of the sterilization treatment
– kd, is the thermal death rate constant
.

Prof. Debabrata Das

Department of Biotechnology
 Kinetics of thermal death of microorganisms:

 On integration of the above equation from t = 0 to t = t, we have the following expression :

Nt/N0 =e-kdt (2)

where
• No is the number of viable organisms present at the start of the sterilization treatment,
• Nt is the number of viable organisms present after a treatment period, t.

On taking natural logarithms, Eq. (2) is reduced to:

ln(Nt/N0) = - kd t (3)

Prof. Debabrata Das

Department of Biotechnology
 Kinetics of thermal death of microorganisms:

The term decimal reduction time, D, is used to characterize the death rate constant.

D is defined as the sterilization time required to reduce the original number of

viable cells by one tenth.

N/N0 = 1/10 = e- kd D

ln (N/No) = - kd D

D = 2.303/kd

Prof. Debabrata Das

Department of Biotechnology
 Plots of the proportion of survivors and the natural logarithm of the proportion of
survivors in a population of microorganisms subjected to a lethal temperature over a
period of time

Prof. Debabrata Das

Department of Biotechnology
This kinetic description makes two predictions which contradict each other

i) An infinite time is required to achieve sterile conditions

ii) After a certain time there will be less than one viable cell remaining

• Thus a value of Nt of less than one organism remaining is considered in terms of

the probability of an organism surviving a treatment

Prof. Debabrata Das

Department of Biotechnology
Typical thermal death rate data for spores of Bacillus stearothermophilus Fs 7954 in distilled
water where N = number of viable spores at any time, N0 = original number of viable spores.
[Adopted from S. Aiba, A.E. Humphrey and N.F. Millis. “Media Sterilization”. In Biochemical
Engineering, 2nd Ed., Academic Press, Inc., New York (1973) 241].

Prof. Debabrata Das

Department of Biotechnology
Typical death rate data for E. coli in buffer, where N = number of viable spores at any time, N0 = original
number of viable spores. [Adopted from S. Aiba, A.E. Humphrey and N.F. Millis. “Media Sterilization”. In
Biochemical Engineering, 2nd Ed., Academic Press, Inc., New York (1973) 241].

Prof. Debabrata Das

Department of Biotechnology
STC on Analysis and Design of Novel Bioreactor, IIT Kharagpur, 1989

Prof. Debabrata Das

Department of Biotechnology
 Two types of sterilization:
Batch sterilization
Sterilizing the entire volume of medium at once using the heating,
holding, and cooling method

Continuous sterilization
Sterilizing only a fraction of the volume at a time by using the medium
as an internal heat exchanger

Prof. Debabrata Das

Department of Biotechnology
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].
Prof. Debabrata Das
Department of Biotechnology
Continuous sterilizer
Continuous medium sterilization is based on the concept of high temperature
short time (HTST) treatment. This takes advantage of the fact that an
increase in temperature has relatively greater effect on thermal destruction of
cells than on nutrients. The steam consumption in continuous sterilization
is perhaps 20 to 25% of the requirements for a batch cycle. The total time
required to sterilize media in case of continuous sterilization is two to three
hours compared to five to six hours in case of batch sterilization.

Prof. Debabrata Das

Department of Biotechnology
HTST treatment for the medium sterilization

Typical Ea values for microorganisms are 250 to 290 kJ mol-1. The

activation energy for vitamins and amino acids is typically 84 to 92
kJ mol-1. This means small increase in temperature has a relatively
greater effect on cell death than on nutrient destruction. This fact
becomes the basis for the use of high temperature short-time (HTST)
sterilization.
STC on Analysis and Design of Novel Bioreactor, IIT Kharagpur, 1989

Prof. Debabrata Das

Department of Biotechnology
 Prof. Debabrata Das
Department of Biotechnology 20