Sterilization

Outline

. Sterilization Methods
. Thermal Death Kinetics
. Design Criterion
. Continuous Sterilization
. Air Sterilization
Sterilization can be defined as the process through which all forms of
life are destroyed removed or permanently inactivated.
Aim
To provide a product that is safe and eliminates the possibility of
contamination
Why
To reduce amount of contaminant's present in environment on surface
of the container closure’s as well as equipment’s and achieve better
sterile condition.
Fermentations are carried out as pure cultures in which only selected
stains are allowed to grow.
If foreign microorganisms exist in the medium or any parts of the
equipment the production organisms have to compete with
contaminants for limited nutrients.
Therefore microorganisms can produce harmful products which can
limit the growth of the production.
Before starting fermentation, the medium and fermentation
equipment have to be free from any living organisms.
 That means they have to be completely sterilized .
Furthermore the aseptic condition has be maintained.
 Sterilization Methods
There are various ways for sterilising fermentation media and equipment.
Heat (moist, dry)
Chemical Agents (like phenols )
Radiation
 Ultraviolet which is non ionization
 X-ray or Gamma-ray is ionization
Mechanical
 Sonic or ultra sonic vibration
Filtration
 high-speed centrifugation, membrane separation
Heat is the most commonly utilised method of sterilisation, and it
may be used
For both liquid medium and heat able solid objects.
It can be used dry or wet (steam).
Moist heat is more effective than dry heat,
In a completely dry state, the inherent heat resistance of vegetative
bacteria cells is substantially strengthened.
As a result death rate is much lower for dry cells than moist.
Heat conduction is also slower in dry air than in steam.
Dry heat is only used to sterilise glassware or heat-able solid items.
The temperature of steam can be raised greatly above the boiling
point of water by pressurising a vessel.
Laboratory autoclaves are generally run at a steam pressure of around
30pisa, which equates to 121oc; at this temperature, even bacterium
spores are quickly killed.
Chemical agents with oxidising or alkylating properties can be
employed to kill bacteria.
They cannot, however, be used to sterilise medium since the
remaining chemical can hinder the fermentation process.
Typically, they are used for disinfection, which is a treatment to
remove or lessen the risk of pathogenic organisms.
Many biological components absorb UV radiation, causing DNA
damage and, as a result, cell death.
The most effective bactericidal wavelength is around 265nm. UV
rays, on the other hand, have a limited ability to permeate matter.
As a result, its application is limited to the reduction of microbial
population in a room where sterility must be maintained.
X-rays are harmful to germs and have the potential to penetrate.
However, due to their high cost and lack of safety, they are ineffective
as sterilisation methods.
High-intensity sonic or ultrasonic waves can disrupt and kill cells.
Rather than sterilisation, the procedure is usually used to disruption
of cells in order to remove intracellular contents.
Filtration is particularly effective for removing microbial from air or
other gases.
In the case of liquid solutions, it is employed with thermolabile
medium or heat-sensitive goods such as human and animal serums
and enzymes.
 Thermal Death Kinetics
First-order kinetics can describe the thermal death of microorganisms
at a specific temperature.
=−

Where is a specific death rate, which is determined not only by

the species but also by the physiological form of cells.
Example
k for bacterial spores at 121oC of the order of 1 min-1
For vegetative cells vary from 10 to 1010 min-1 depending on the particular
organisms.
 ln = −∫

= −∫
Shows the exponential decay of cell population. The temperature
dependence of the specific death rate kd can be assumed to follow the
Arrhenius equation.

Where Ed is activation energy which can be obtained from the slop of

ln(Kd) versus 1/T plot
 Design Criterion
From Equation
ln = −∫ and

The design criterion for sterilization can be defined as (Deindorefer and

Humphrey, 1959)

Which is also known Del factor, a measure of the size of the

job to be accomplished.
The Del factor increase as the final number of cells decrease.
For Example the Del factor to reduce the number of cell in the
fermenter from1010 viable organisms to one is
10
∇= ln = 23
1
Theoretically impossible to ensure total destruction of viable cells
Therefore it is expressed as the fraction of one, is the probability of
contamination
Example n=0.001 in this fraction the chance of surviving is 1 is a 1000
Del factor to reduce the number of cells in a fermenter form 1010 viable
to 0.001 is
∇= ln = 30
.
 Batch Sterilization
In a fermenter, medium sterilisation can be done in batch mode
using
Direct steam sparging,
Electrical heaters, or
Circulating constant pressure condensing steam through a heating
coil.
Sterilization have three cycles
Heating
Holding and
Cooling
Total Del factor is the sum of three cycle Del factor
∇ =∇ +∇ +∇
Values of ∇ and ∇ can be determined by the methods used for
heating and cooling
∇ is determined by the length of the controlled holding period
The following are Design procedure for the estimation of holding time
I. Calculate the total sterilization criterion
II. Measure the temperature versus time profile during heating holding and
cooling time of sterilization .
If experimental measurement are not practical, theoretical
equation for heating and cooling can be employed
a. For batch heating direct steam sparging

b. For batch heating with constant rate of heat flow like electrical
heating.
C. Batch heating isothermal source

d. Batch cooling using a continuous non-isothermal heat sink such

as passing cooling water through cooling coil
3. plot the value of Kd as a function of time
4. integrate the area under the Kd versus time curve for heating and
cooling periods. The holding time can be calculated
∇
=
 Continuous Sterilization
Sterilization can be carried out in a continuous mode also
Continuous sterilization have several advantages.
I. Simplifies production.
II. Provides reproducible conditions.
III. Can be operated at high temp. (140oC) and less holding time 1-2
minuets.
IV. Required less steam since it can recover heat from the sterilized
medium.
V. Easier to automate the process.
Continuous sterilization have three main section heating, holding and
cooling.
The temperature change with respect to residence time ̅ as the
medium pass through an isothermal heat source can be approximated as
(Deindoerfer and Humphrey 1959b)

Countercurrent heat source of equal flow rare and heat capacity

Holding section: Heating medium passes through a holding section
which is usually composed of long tube.
The holding section is maintained in adiabatic condition

For Del factor

no is the number of cells at the beginning of the holding section

If the medium in a holding section behaves as an ideal plug flow, the
medium in the section will have the same residence time for all
medium.
The average fluid velocity to maximum velocity ratio for laminar
flow of Newtonian fluids through a smooth round pipe is 0.5.
The ratio rapidly changes from 0.5 to around 0.75. When the laminar
flow transitions to turbulent, the value eventually rises to 0.87.
When Reynold’s number is about 106 (McCable .et.al 1985)
As a result, if we use the mean velocity to calculate the needed residence time for
sterilisation.
Some of the medium will be under sterilized, potentially causing a major
contamination problem.
Material balance for the microorganisms in the medium
In-Out-Killed by Sterilization =Accumulation
At steady state, accumulation term is zero
Input and output of the microorganisms into or out of the element
have both a bulk flow and axial diffusion condition.
The number of microorganisms entering minus those leaving by bulk
flow is
 Cooling Section
A quench cooler with adequate heat removal capacity is effective.
Another technique is to inject the hot medium through an expansion
value into vacuum chamber. Which known as flash cooling
Temperature versus residence time relationship for cooling using an
isothermal heat sink is.

Cooling countercurrent heat sink of equal flow rate and heat capacity
 Air Sterilization
Aerobic fermentation air needs to be supplied continuously.
Aeration rates for aerobic fermentation are 0.5-1vvm (air volume per
liquid per minute)
Air must be free of microbial contaminants
Sterilizing air in heat is not economical and also ineffective due to
low heat transfer efficiency of air compared with liquid
The most effective technique for air sterilization is filtration using
fibrous or membrane filters.
Fibrous filters airborne particles are collected by the following
mechanisms
Impaction
Interception
Diffusion
Example
Steam injector and flash cooler is used to sterilize medium with flow
rate of 2m3/hr. bacteria count of medium is 5*1012m-3. needs to
reduced only one microorganisms can survive during two month of
continuous sterilization , spore in the medium can be characterized by
Arrhenius coefficient Kdo 5.7*1039hr-1and activation energy Ed
2.834*105 kJ/kmol. The sterilize have inner diameter 0.102m steam at
600kpa(Gage pressure) and operating temp. 125oC.
The physical properties of the medium at 125oC are, C= 4.187KJ/Kg
k, density 1g/cm3
=4 .ℎ
I. What length should the pipe be in the sterilizer if you
assume ideal plug flow?
 Chapter 8

Agitation and Aeration

Outline
Basic Mass-Transfer Concepts
Correlation for Mass-Transfer Coefficient
Shear-Sensitive Mixing
 Introduction
One of the most important factors to consider in designing a
fermenter is the provision for adequate mixing of its contents.
The main objectives of mixing in fermentation are
 Disperse the air bubbles
 Suspend the microorganisms
 Enhance heat and mass transfer in the medium.
Most nutrients are highly soluble in water very little or limited
mixing is required during fermentation.
Dissolved oxygen solubility in a fermentation medium is very low but
demand for the growth of aerobic microorganisms are high
Maximum concentration of oxygen in aqueous solution is on the
order of 6-8mg/L.
Oxygen requirements for cells although it can vary widely depending
on microorganisms on the order of 1g/L.
Adequate oxygen supply to cells is often critical in aerobic
fermentation.
Laboratory shaker apparatus is adequate to cultivate microorganisms
Mass-Transfer Path
The path of gaseous substrate from gas bubble to an organelle in
microorganism can be divided in to several steps.
1. Transfer from bulk gas in a bubble to relatively unmixed gas layer.
2. Diffusion through the relatively unmixed gas layer
3. Diffusion through the relatively unmixed liquid layer
surrounding the bubble.
4. Transfer from the relatively unmixed liquid layer to the bulk
liquid.
5. Transfer from the bulk liquid to relatively unmixed liquid
layer surrounding a microorganism.
6. Diffusion through the relatively unmixed liquid layer.
7. Diffusion from the surface of microorganism to organelle in
which oxygen is consumed.
 Mass-Transfer Concepts
Molecular Diffusion in Liquids
When the conc. of a component varies from one point to another,
components has tendency to flow in the direction that will reduce the
local difference in conc.
Molar flux of a component A relative to average molar velocity of all
constituent JA is proportional to the concentration gradient dCA/dz

Fick’s first law for z-direction

Molar flux relative to stationary coordinates NA is equal to

Where
◦ C: Total concentration of component A and B
◦ NB: is the molar flux relative to stationary coordinate
For Dilute solution of A
 Diffusivity
Kinetic theory of liquids is much less advanced than that of gases.
Therefore, the correlation for diffusivities in liquid is not as reliable
as that of gas
Among several correlation the Wilke-Chang correlation is the most
widely used for dilute solution of nonelectrolytes
When the solvent is water Othmer and Thakar Correlation
Example
Estimate the Diffusivity for oxygen in water at 25oCcompare the
prediction from the Wilke-Chang and Othmer-Thakar correlation
with the experimental values of 2.5*10-9m2/s. convert the
experimental value to that corresponding to a temperature of 40oC
 Mass-Transfer Coefficient
The mass flux, the rate of mass transfer qD per unit area is proportional
to a concentration difference
If a solute transfer from the gas to the liquid phase its mass flux from
the gas phase to the interface NG is

Where
CG and CGi is the gas-side conc. at the bulk and interface respectively
KG is the individual mass-transfer coefficient for the gas phase and
A is the interfacial area
Similarly, the liquid phase-side phase mass flux NL is
Where
KL is the individual mass-transfer coefficient for the liquid phase
 Mechanism of Mass-Transfer
Different mechanisms have been proposed to provide a basis for
theory of interphase mass-transfer
The three best known theories are
Two-film theory
Penetration theory
Surface renewal theory
 Two-film Theory
Assume that the entire resistance to transfer is contained in two
fictitious films on ether side of the interface in which transfer occurs
by molecular diffusion.
This model leads to the conclusion that the mass-transfer coefficient
KL is proportional to diffusivity DAB and inversely proportional to the
film thickness Zf
 Penetration Theory
Assumes that turbulent eddies travel from the bulk of the phase to the
interface where theory remain for constant exposure time te.
The solute is assumed to penetrate in to a given eddy during its stay at
the interface by process of unsteady-state molecular diffusion
The model predicts that the mass-transfer coefficient is directly
proportional to the square root of molecular diffusivity.
 Surface Renewal Theory
Proposes that there is an infinite range of ages for element of the
surface and the surface age distribution function.

Where s: is the fractional rate of surface renewal.

This theory also predicts mass-transfer coefficient is proportional to
the square root of the molecular diffusivity.
Correlation for Mass-Transfer Coefficient
The mass-transfer coefficient depends on the geometry of the vessel and its
physical characteristics.
Deriving a useful connection from a merely theoretical basis is challenging,
if not impossible, due to the complexity of hydrodynamics in multiphase
mixing.
Fitting experimental data to produce an empirical correlation for the mass-
transfer coefficient is a frequent method.
Since dimensionless groups are helpful for scale-up procedures and are
dimensionally consistent, they are typically used to describe correlations.
The group without dimensions vital for correlations can be obtained with
the application of Buckingham Pi theory.
 Shear-Sensitive Mixing
The mechanically agitated fermenter is one of the most adaptable
fermenter systems utilised in industry.
This kind of technology works well for blending the contents of the
fermenter, suspending the cells, fracturing air bubbles to improve
oxygenation, and preventing the formation of big cell aggregates.
Nonetheless, the agitator's shear can damage some microorganisms'
cell membranes and ultimately cause their death.
Shear also responsible for the deactivation of Enzymes.
In order to ensure that an agitated fermentation system operates as
efficiently as possible, we must comprehend the hydrodynamics of
shear-sensitive mixing.
For the laminar flow region of Newtonian

For turbulent flow

where η is the eddy viscosity, which depends on the operating
conditions as well as the fluid's physical characteristics.
It is therefore simpler to estimate shear rate du/dy rather than shear
stress in order to characterise the intensity of shear in a turbulent
system, such as an agitated fermenter.
Although the shear rate serves as a gauge for shear severity, it is
important to keep in mind that shear stress is what ultimately damages
live cells or enzymes.
Depending upon the magnitude of viscosity and also whether the flow
is laminar or turbulent, there is a wide range of shear stress generated
for the same shear rate
However shear rate is a good measure for the intensity of agitation.
The complex fluid dynamics produced by impellers make it difficult, if
not impossible, to calculate the shear rate in agitated systems.
When a fluid element travels through the impeller region, it
experiences maximum shear, and when it passes close to the vessel's
corner, it experiences minimal shear.
This is the wide range of shear rates that a fluid element experiences
during agitation.
Metzner and Otto (1957) developed a general correlation for the
average shear rate generated by a flat-blade disk turbine based on
power measurement on non-Newtonian liquids
 Chapter 9

Downstream Processing
 Outline
In this section we will see in brief
Solid-Liquid Separation
Cell Rupture
Recovery and Purification
Purification and separation of the desired products are necessary following an
effective fermentation or enzyme reaction.
This last stage, sometimes referred to as downstream processing or bio
separation, can make up as much as 60% of the entire production costs, not
including the price of the raw ingredients that were obtained.
The fermentation products can be
The cells (biomass)
Components within the fermentation broth (extracellular)
Trapped in Cell (intracellular)
If the cell is the product of interest, it is isolated from the
fermentation broth and cleaned and dried.
After the cells are separated, the extracellular products in the dilute
aqueous medium must be collected and purified.
The intracellular products can be collected and purified after the cells
have been ruptured.
The downstream processing for enzyme reactions will be identical to
the extracellular product processing.
 Bio separation process make use of many separation techniques commonly used
in the chemical process industries.
However, bio separation have distinct characteristic that are not common in the
traditional separation of chemical processes.
I. The products are in dilute concentration in an aqueous medium
II. Usually temperature sensitive
III. Great verities of product to be separated
IV. Can be intracellular often insoluble inclusion bodies
V. Physical and chemical properties of products and contaminants are similar
VI. Extremely high purity and homogeneity are demand for human health care
 Solid-Liquid Separation
The first step in downstream processing is the separation of insoluble from
the fermentation broth.
The selection of separation technique depends on the characteristics of
solid and liquid medium
The solid particles to be separated are mainly cellular mass with specific
gravity of about 1.05-1.1
Shapes of the particles maybe spheres, ellipsoids, rods, filaments or
flocculants.
Separation of solid particles from the fermentation broth can be
accomplished by filtration or centrifugation.
 Filtration
Filtration separates particles by forcing the fluid through a filtering
medium on which solid are deposited.
Filtration can be divided into several categories depending on the
filtering medium used, the range of particle size removed, the
pressure difference and principle of filtration
Such as
Conventional filtration
Microfiltration
Ultrafiltration
Revers osmosis
The two type of filter most used for cell recovery are the filter press
and rotary drum filters
Filter press is often employed for the small-scale separation of
bacteria and fungi from the broth.
Rotary drum filter are usually used for large scale filtration.
Common filter medium is cloth filter made of canvas, wool, synthetic
fabrics, metal or glass fiber
 Centrifugation
Centrifugation is an alternative method when the filtration is effective
like in the case of small particles size.
It requires more expensive equipment than filtration.
Cannot be scaled to the same capacity as filtration equipment.
The two basic types of large-scale centrifuge are tubular and dick
centrifuge.
Tubular Centrifuge consists of a hollow cylindrical rotating element
in a stationary casing.
The suspension is usually feed through the bottom and clarified liquid
is removed from the top leaving solid deposit on the bowl’s wall
Accumulated solids are recovered manually from the bowl.
A typical tubular centrifuge has a bowl diameter of 2-5in and height
9-30in with maximum rotating speed of 15,000 to 50,000rpm.
Disk Centrifuge mostly used for bio-separation
It has the advantage of continuous operation
It consists of short and wide bowl 8-20in runs in vertical axis.
Liquid enters at the bottom flows into the channels and upward past
the disk
Solid particles are thrown outward and clear liquid flows towards the
center of the bowl and discharge through an annular slit.
The collected solids can be removed intermittently or continuously.
 Cell Rupture
Once the cellular materials have been separated, those containing
intracellular proteins must be disrupted in order to release their
products.
Cellular materials are typically difficult to disrupt due to the strength
of the cell walls and the strong osmotic pressure inside.
Cell rupture procedures must be extremely forceful, yet they must
also be gentle enough not to destroy desired components.
Physical, chemical, or biological approaches can be used to break
cells.
Physical methods include disruption by milling, homogenization or
ultrasonication
High speed bead mill are composed of a grinding chamber filled with
glass or steel beads which are agitated with disk or impellers.
The efficiency of cell disruption in bed mill depends on
Concentration of the cell
Amount and size of beads
Type and rotation speed of agitators
 High-pressure Homogenizer
A positive displacement pump with an adjustable orifice value.
Is the most widely used methods for large scale cell disruption
The pump pressurize the cell suspension to approximately 400-
500bar and rapidly release it through a special discharge valve
This create very high share rate.
In order to compensate for heat generated during adiabatic
compression the system is refrigerated to 4-5oC
 Ultrasonicate
An ultrasonicate produces sound waves at frequencies more than 16
kHz, causing pressure variations to form oscillating bubbles that
rupture rapidly, generating shock waves.
Ultrasonic cell disruption is successful with most cell suspensions
and is commonly employed in the laboratory.
However, due to its high running costs, it is impossible to utilise on a
broad scale.
 Chemical Methods
Chemical cell rupture methods include treating cells with
detergents (surfactants),
alkalis,
organic solvents, or
osmotic shock.
Chemical procedures necessitate that the product be resistant to the
harsh environment created by the chemicals.
Following cell disruption, the chemicals must be easily separable or
compatible with the products.
Surfactants break the cell wall by dissolving the lipids within it.
Surfactants commonly used in laboratories include sodium
dodecylsulfate (SDS), sodium sulfonate, Triton X-IOO, and sodium
taurocholate.
The saponification of lipids is one of the ways that alkali treatment
destroys cell membranes.
Although alkali treatment is affordable and effective, it is so severe
that the protein products may be denatured.
Organic solvents, such as toluene, can also cause cell wall rupture by
entering the cell wall lipids and expanding the wall.
When red blood cells or other animal cells are dropped into pure
water, they can swell and burst due to the osmotic flow of water into
the cells.
 Biological Methods
Enzymatic cell wall digesting is a good example of biological cell
disintegration.
It is an efficient process that is also selective and mild,
but its high cost makes it unsuitable for large-scale operations.
 Recovery and Purification
Recovery
After separating solid and liquid (and disrupting cells in the case of
intracellular products),
we have a dilute aqueous solution from which products must be
recovered (or concentrated) and purified.
Recovery and purification are indistinguishable since both use the
same processes.
However, among the several separation techniques utilised in the
recovery step, extraction and adsorption are widely used as recovery
methods.
 Extraction
Extraction is a separation process that separating the constituents of
(solutes) of the liquid solution by contact with another insoluble
liquid or solvent.
During the liquid-liquid contact the solvents will be distributed
differently b/n the two liquid phases.
By choosing a suitable solvent the desired product can be selectively
extracted.
After the extraction is completed the solvent rich phase is called
extract and the residual liquid is raffinate.
Single-Stage Extraction
Multistage Extraction
 Adsorption
A specific substance in solution can be adsorbed selectively by
certain solids as a result of either physical or chemical interactions
b/n the molecules of the solids and substance adsorbed.
Since the adsorption is very selective while the solute loading on the
solid surface is limited, adsorption is an effective methods for
separation of very dilute dissolved substance.
Adsorption can be classified in to three ways
 Conventional Adsorption
 Ion exchange
 Affinity Adsorption
 Conventional Adsorption
Adsorption is a reversible process caused by intermolecular forces of
attraction (van der Waals forces) between solid molecules and the
substance adsorbed.
Activated carbons are the most commonly utilised adsorbents in bio-
separations among the many available in industry.
They are frequently used to remove trace amounts of contaminants
from potable water or processing liquids.
They are also utilised to isolate valuable by products from
fermentation broth via adsorption and subsequently recovery via
elution.
 Ion-exchange
An ion-exchange resin is made up of three basic components: a polymeric
network (such as styrene-divmyl-benzene, acrylate, methacrylate,
plyamine, cellulose, or dextran),
ionic functional groups (which might be anions or cations), and
counterions.
Strong-acid cation-exchange resins, for example, have fixed charges such
as –SO3-, which are created by sulfonating the polymer's benzene rings.
To preserve electrical neutrality, fixed ionic sites in the resin are balanced
by an equal number of charged ions with opposite charge (counterions).
When a solution containing positively charged ions comes into contact with
cation-exchange resins,
Positively charged ions replace the counter ions and are removed from the
solution, which is the principle of ion-exchange separation.
Consider the reaction of the hydrogen form of a cation-exchange resin (H+R-)
with sodium hydroxide to demonstrate the ion-exchange principle.
 Affinity Adsorption
Affinity Adsorption is based on the chemical interaction between a
solute and ligand which is attached to the surface of the carrier
particle by covalent or ionic bond.
It offers high selectivity in many bio-separation.
However, the high cost of the resin is a major disadvantage and limits
its industrial use.
 Purification
Following the recovery or isolation of a product, it may need to be
purified further.
Many methods of purification exist, including
 Precipitation,
 Chromatography,
 Electrophoresis, and
 Ultrafiltration.
 Precipitation
Precipitation is commonly employed to recover proteins or antibiotics. The
addition of salts, organic solvents, or heat can cause it.
The addition of salt precipitates proteins because the increase in salt
concentration in solution reduces protein solubility significantly.
Precipitation is both efficient and affordable.
It results in minimal denaturation.
The most often used salt is ammonium sulphate. Ammonium sulphate has
the disadvantage of being difficult to extract from precipitated protein.
 Chromatograph
Chromatograph processes always involve a mobile phase and a
stationary phase.
The mobile phase is the solution containing the solute to be separated
and eluent that carries the solution through the stationary phase.
The stationary phase can be Adsorbent, ion-exchange resin, porous
solid or gel which are usually packed in the cylinder column.
 Electrophoresis
When a mixture of solute is placed in an electrical field the positively
charged species are attracted to the anode and the negatively charged ones
to the cathode.
The separation of charged species based on the specific migration rates in
an electrical field is electrophoresis.
It is one the most effective methods of protein separation and
characterization.
It can be performed under very mild condition and it has high resolving
power in a clear separation of similarly charged protein molecules.
In order to use the components of the mixture must have an ionic
form and each components must posses a different net charge.
Electrophoresis is performed in gel, known as gel electrophoresis.
Polyacrylamide gels are most commonly used because of it is
thermostable, transparent, durable, relatively inert chemically, and
easily prepared.
Gel chromatography is that setting up the gel is tedious and time
consuming and large-scale operation is not possible.
 Membrane Separation
We can use filtration principle for separation of small particles down
to small size of molecular level by using polymeric membrane.
Depending upon the size range of the particles separated, membrane
separation processes can be classified in to the following

 Microfiltration
 Ultrafiltration
 Reverse Osmosis