Medium sterilization

Why do we need it?

• Medium sterilization is one of the most the one of the first and most critical unit operation required for successful
fermentation.
• The objective is to prevent the growth of undesired microorganism during the course of a fermentation and
bioconversion enzyme catalyzed reaction and medium storage

Media can be sterilized by

1) Filtration,
2) Radiation,
3) Ultrasonic treatment,
4) Chemical treatment
5) Heat

Out of these methods, heat or steam is the most useful method for the sterilization of fermentation media
Heat Sterilization

• Dry heat - it requires higher temperature in the range of 160 to 180 degree centigrade. And required the exposure time 2
hours depending upon the that temperature employment. Some items like, inoculating loops, which are used routinely in
the laboratory, can be sterilized in a small, bench-top incinerator. Dry air heat is less effective than moist heat. The spores
of Clostridium botulinum, the cause of botulism, are killed in 5 minutes at 121°C by moist heat but only after 2 hours at
160°C with dry heat. However, dry heat has some definite advantages. It does not corrode glassware and metal
instruments as moist heat does, and it can be used to sterilize powders, oils, and similar items. Most laboratories sterilize
glassware and pipettes with dry heat.
• Moist heat readily kills viruses, bacteria, and fungi. In order to destroy bacterial endospores, moist heat sterilization must
be carried out at temperatures above 100°C, and this requires the use of saturated steam under pressure. Steam
sterilization is carried out with an autoclave. The desired temperature and pressure, usually 121°C and 15 pounds of
pressure. At this temperature saturated steam destroys all vegetative cells and endospores in a small volume of liquid
within 10 to 12 minutes. Treatment is continued for at least 15 minutes to provide a margin of safety. Of course, larger
containers of liquid such as flasks and carboys require much longer treatment times.
• Thermal death time (TDT) is the shortest time needed to kill all organisms in a microbial suspension at a specific
temperature and under defined conditions. However, such destruction is logarithmic, and it is theoretically not possible to
completely destroy microorganisms in a sample, even with extended heating.

• Decimal reduction time (D) or D value is the time required to kill 90% of the microorganisms or spores in a sample at a
specified temperature or sterilization time required to reduce the original number of viable cells by one tenth.

Kinetics of thermal death rate of microorganism - dN/dt = kd N

On integration,
ln (Nt / N0) = - kd t
Nt/N0 = e- kdt
ln (1 / 10) = - kd D
kd is the thermal death rate constant,
D = 2.303/ kd
N is the number of viable organisms
N0 – initial number Or you can say Decimal reduction time (D) is inversely proportional to k d.

Nt – number at time t Which means with increase in temperature increases, kd value also increases. Now

t is sterilization treatment time as your kd value increases then thermal decimal reduction time will reduce significantly
 So, what will be the slope here?

This kinetic description makes two predictions, which appear abnormal.

1. Sterile condition is achieved in an infinite time (i.e. Nt = 0)
2. After a definite time number of cells present will be less than one, that means getting 100 percent sterility is very
difficult to achieve
 • A plot of ln N(t)/N0 versus time is called a survival curve.
• Such a curve is implicitly deterministic.
• Extrapolation of a survival curve to one organism or less is not proper. Most
real populations are not homogeneous. Often a subgroup is more resistant
to the sterilization agent. Also, organisms growing in clumps tend to be more
kd value resistant to death. These factors are important in processing
increases as
the temperature
increases – requires
less time to kill

a) Typical death-rate data for spores of

Bacillus stearothermophilus.
(b) Typical death rate for E. coli in buffer
In Fig. 5.3a the population consists mainly of the less-
Figure 5.3b represents the reverse situation where the more
resistant type where the initial decline is due principally
resistant type predominates and its presence disguises the
to the destruction of the less-resistant cell population
decrease in the number of the less resistant type
and the later, less rapid decline, is due principally to the
destruction of the more resistant cell population
• Which means Time for the sterilization is also dependent on the type of the population. If the sensitive organisms are
more in number than whole culture sterilization will be equal to that of the sensitive culture. However, if the number of
the resistant organisms is more than the sensitive organisms of whole culture is equal to that of the sterilization of the
resistant organisms. Now by considering that contaminant may be by not a single type of organism but by different
types of organisms. Sterilization of media required destruction of all types of organisms.

• Depending on the rate of heat transfer from the steam or electrical element, raising the temperature of the medium in
large fermenters can take a significant period of time. Once the holding or sterilisation temperature is reached, the
temperature is held constant for a period of time thd. Cooling water in the coils or jacket of the fermenter is then used
to reduce the medium temperature to the required value.
• For operation of batch sterilisation systems, we must be able to estimate the holding time required to achieve the
desired level of cell destruction. As well as destroying contaminant organisms, heat sterilisation also destroys nutrients in
the medium. To minimise this loss, holding times at the highest sterilisation temperature should be kept as short
as possible. You can also call it HTST – High Temp Short Time
• Cell death occurs at all times during batch sterilisation, including the heating-up and cooling-down periods. The holding
time thd can be minimised by taking into account cell destruction during these periods.
(a) Variation of temperature with time for batch sterilisation of
(b) Reduction in number of viable cells during batch
liquid medium.
Cell numbers decreasing in every sterilisation.
time periods i.e. , including the
heating-up, holding or sterilisation The holding time can be given as:
temperature, and cooling-down
period

where thd is the holding time,

N1 is the number of viable cells at the start of holding,

N2 is the number of viable cells at the end of holding

 As you can see, Kd depends on Temperature. Hence kd can be evaluated as a function of Temperature using Arrhenius
equation:

where A is the Arrhenius constant or frequency factor,

Ed is the activation energy for the thermal cell death,
R is the ideal gas constant and T is absolute temperature

Combining eqn - dN/dt = kd N , we can get

Integration of this Eq. gives for the heating period:

And for the cooling period:

where t1 is the time at the end of heating, t2 is the time at the end of holding,

t is the time at the end of cooling.

Drawbacks of batch sterilization
• In case of batch sterilization process, if you move from lab scale to the commercial scale you have to handle huge
amount of liquid. Naturally you have that the time requirement for the sterilization very high as compared to small
volume – in short During the scale up the longer treatment time are required to achieve the same sterilization

• Second bottleneck - Sustained the elevated temperature during the heating and cooling damage the vitamins,
proteins and sugar which reduce the quality of the nutrient in the medium.

• Longer holding time is required to treat the solid phase substrate and media containing the particles

• Large amount of volume is used to make the batch process tedious and energy intensive

These are the reasons we do not use the batch

sterilization process in the industry for the sterilizing the
media.
 Continuous sterilization

• Continuous sterilization based on the concept high-temperature, short-exposure-time process (HTST)

• It can significantly reduce the damage to medium ingredients while achieving high levels of cell destruction.
• Other advantages include improved steam economy and more reliable scale-up. Steam consumption is reduced to
20 to 25 % that used in batch processes
• The time required is also significantly reduced because heating and cooling are virtually instantaneous - that total
time required for the sterilization of the continuous sterilization system is 2 to 3 hours as compared to 5 to 6 hours in
case of batch sterilization.

Methods of Continuous sterilization

i. Direct steam injection

ii. Plate heat exchanger

Direct steam injection

• In this process Rapid heat up of medium takes place without the use of
heat exchanger
• Particularly effective with the media that tends to foul heat exchanger
• Disadvantage is the dilution of the media with condensed steam which
makes it difficult to control pressure and temperature due to variation of
viscosity – so what exactly happens? 80°C 140°C
37°C
as soon as you mix together the steam with medium, steam will
undergo condensation, as soon as the steam undergo condensation
your media will be diluted and viscosity will be changed, density will
be changed, we know the flow characteristics of the fluid
depends on the viscosity and the density. So, as they all are
changing. – finally the flow characteristics will change and
temperature control will be a problem
37°C
 Medium and steam mixing is happening here.
Direct steam injection So, the main advantage of this process is that raw
media and steam they instantaneously mix together
and raise the temperature.
The vapor will go out. You might have seen
140°C that lot of industry they are discharging lot of white
flues and white flues are nothing, but it is vapor that is
coming out from the industry

Flash cooling will

take place
This is the Holding section and this is pipe is totally
insulated so, that no heat loss take place.
The sterilization temperature is 140, because the
industry use 140 degree centigrade, to keep it for So, the temperature instantaneously
increase 140 degree centigrade and
very short time not like our lab 121 degree hold it and
centigrade. So, this is the rapid heat up of the media then flash cooling to 80 degree
centigrade,
that take place then slowly cooled down to 37 degree
centigrade.
Plate heat exchanger
• In this process, the raw medium entering is first pre-heated (I) by hot sterile medium in heat exchange.
• This economies on stream requirement for heating the raw Heat exchanger or
Hot sterile medium entering – so
economizer
and cooling the sterile medium sterile medium cooling down
37°C 80°C 140°C - Heat is being
• Drawbacks - Fouling of hot heat exchange surface and the exchanged
between cold raw
leak around exchanger gaskets and seals 140°C and hot sterile
• Plate heat exchanger is mostly used by the industry (III) (I) medium - so we
(II)
can save lot of
energy and that is
80°C why we call it
140°C economy

37°C
raw medium entering – so
raw medium becoming pre-heated
 Design of continuous sterilizer

• An important variable affecting performance of continuous sterilizers is the nature of fluid flow in the system. Ideally, all
fluid entering the equipment at a particular instant should spend the same time in the sterilizer and exit the system
at the same time; unless this occurs we cannot fully control the time spent in the sterilizer by all fluid elements.
• The flow is somewhere between fully viscous and fully turbulent flow

• The type of flow in pipes where there is neither mixing nor variation in fluid velocity is
called plug flow. Usually we prefer plug flow which is the ideal flow pattern, but very difficult to
achieve. In reality, fluid elements in pipes have a range of different velocities. And we usually
use the turbulent flow. In order to maximize the overheating of the media it is desirable to
approach closely fully turbulent flow. In fully developed turbulent flow, the velocity distribution
approaches that of plug flow; however there is some reduction of flow speed at the walls

• Laminar flow you can see the velocity gradient act across the cross section of the tube.

Less time for sterilization and underheating

overheating
• Deviation from plug-flow behavior is characterized by the degree of axial dispersion in the system, i.e. the degree to
which mixing occurs along the length or axis of the pipe.
• Axial dispersion is a critical factor affecting design of continuous sterilizers.

• The relative importance of axial dispersion and bulk flow in transfer of material through the pipe is represented
by a dimensionless variable called the Peclet number where Pe is the Peclet number,
u is the average linear fluid velocity,
L is the pipe length
Dz is the axial-dispersion coefficient

• For perfect plug flow, Dz is zero and Pe is infinitely large;

• In practice, Peclet numbers between 3 and 600 are typical.
• The value of Dz for a particular system depends on the
Reynolds number and pipe geometry.
Reynolds Number
• The Reynolds number is the ratio of inertial forces to
viscous forces and is a convenient parameter for predicting
if a flow condition will be laminar or turbulent.
• It can be interpreted that when the viscous forces are
dominant (slow flow, low Re) they are sufficient enough to
keep all the fluid particles in line, then the flow is laminar.
• When the inertial forces dominate over the viscous forces
(when the fluid is flowing faster and Re is larger) then the
flow is turbulent.

• It is a dimensionless number comprised of the physical

characteristics of the flow. An increasing Reynolds number
indicates an increasing turbulence of flow.
• A correlation from the engineering literature for evaluating D z is shown in Figure

• Once the Peclet number has been calculated from Eq. (13.101), the extent of
cell destruction in the sterilizer can be related to the specific death constant k d
using the Figure or graph.
• In Figure, N1 is the number of viable cells entering the system, N 2 is the
number of cells leaving, Pe is the Peclet number as defined by Eq. (13.101),
and Da is another dimensionless number called the Damkohler number:

where kd is the specific death constant,

u is the average linear fluid velocity,
L is the pipe length So, if you know N2/N1, you can
easily get Da from the graph.
• The lower the value of N2/N1 , the greater is the level of cell destruction. Thus by using Da the length of
the sterilizer can be calculated
Problem

Medium at a flow rate of 2 m3 h-1 is to be sterilised by heat exchange with steam in a continuous steriliser. The liquid
contains bacterial spores at a concentration of 5*1012 m-3; the activation energy and Arrhenius constant for thermal
destruction of these contaminants are 283 kJ g mol-1 and 5.7 x 1039 h-1, respectively. A contamination risk of one
organism surviving every 60 days' operation is considered acceptable. The steriliser pipe has an inner diameter of 0.1 m;
the length of the holding section is 24 m. The density of the medium is 1000 kg m -3 and the viscosity is 3.6 kg m-1 h-1. What
sterilizing temperature is required?

A contamination risk of one organism surviving every 60 days'

operation is considered acceptable –
Which means one organism surviving every 60 days operation or you can
say in every 60 days operation, we are allowing one organism to pass
through –
 0.1
πr 2

Re – Reynold’s number
D – pipe diameter
u – fluid velocity
For Re= 7.07 x 103 we can determine Dz from Figure 13.40 using the experimental curve (this µ - viscosity
data/graph is generally given or obtained from experiments) ρ - density
The sterilisation temperature can be evaluated after rearranging Eq.

Da = 42
 Air sterilization

• The main purpose of air sterilization is to remove of air borne of microorganism present in the air, for aerobic
fermentation process.

• There are wide variation and the quantity of suspended particles and microbes present in the atmospheric outdoor

• The microorganism may be vary in the range 10 - 2000 /m3 while the suspended particle may vary 22 - 10000/m 3

• Among the microorganisms – fungal spores (50 %) and gram negative bacteria (40%) that dominated
Methods of air sterilization

• Filtration
• Heat
• Ultraviolet rays or electromagnetic waves
• Chemical agent

• Among this industrially the heat and filtration are the most commonly used;

• However, heat is ineffective in case of air sterilization due to low heat transfer efficiency of air as compared
to liquid

• Thus more effective technique of air sterilization is filtration with fibrous and membrane filter
Filtration
• Filtration is an excellent way to reduce the microbial population in solutions of heat-sensitive material, and sometimes it can
be used to sterilize solutions.
• Filtration process does not destroy, but simply remove the microorganism. It is used both for the clarification and
sterilization of the liquid as it is capable of preventing the passage of both viable and non-viable particles.
• Membrane filters are porous membranes, made of cellulose acetate, cellulose nitrate, polycarbonate, polyvinylidene
fluoride, or other synthetic materials.
• Although a wide variety of pore sizes are available, membranes with pores about 0.2 µm in diameter are used to remove
most vegetative cells, from solutions ranging in volume from 1 ml to many liters.
• These filters are used to sterilize pharmaceuticals, ophthalmic solutions, culture media, oils, antibiotics, and other heat-
sensitive solutions.
• Air also can be sterilized by filtration. Common examples are surgical masks and cotton plugs on culture vessels that let air in
but keep microorganisms out. Other important examples are laminar flow biological safety cabinets, which employ high-
efficiency particulate air (HEPA) filters (a type of depth filter) to remove 99.97% of 0.3 µm particles. Laminar flow biological
safety cabinets or hoods force air through HEPA filters, then project a vertical curtain of sterile air across the cabinet opening.
This protects a worker from microorganisms being handled within the cabinet and prevents contamination of the room
• Polytetrafluoroethylene (PTFE) membranes are compatible with almost all solvents, acids and bases. Main
applications are the filtration of non-aqueous sample
• Nylon 66 membranes are compatible with most solvents, organic and aqueous; but use with strong acids,
methylene chloride and DMF is not recommended.
• Polyethersulfone (PES) Hydrophilic membrane. Broad solvent compatibility. Suitable for filtration of
aqueous and compatible organic solvents. Higher liquid flow than either PTFE or PVDF. Low in extractables.
Low protein binding.
• Cellulose nitrate is primarily used for pre-filters and is compatible with many but not all aqueous or non-
aqueous solvents. High protein binding capacity, which makes it unsuitable for protein recovery applications
• Cellulose acetate membranes are not compatible with organic solvents. They are well suited for aqueous
solutions. Very low protein binding capacity, which makes it an excellent choice for protein recovery
applications
What is your desired end product?

• Interested in particles retained on the membrane surface

i. Polycarbonate (PCTE) and PETE for easy surface retrieval/Microscopy,
ii. Mixed cellulose ester (MCE) for retention and plating of bacteria from solution

• Interested in the filtered fluid

i. MCE and Nylon have high protein-binding capacity for sterilization
ii. Cellulose Acetate, Polyethersulfone (PES), polypropylene (PP), and PVDF have good flow rates
iii. PAN is designed for fast water purification
Selection criteria of the air filter

• Filter retention efficiency – means how much organism it can retain and for how long; that means, how long this filter
can be utilized
• Economy – cost effective or not?
• Ease to use

• Most important selection criteria of the filter is efficiency and reliability of the organism Retention
• Fix submicron pore size membrane filter provides the highest level of filtration efficiency.

• Membrane is usually polymeric material and importantly Membrane is a very weak material.
• So, as your pore size decreases, the pressure drop across the membrane will be very high. If the pressure drop is
very high there is a every possibility that busting of the membrane material. So, life of the membrane reduces
significantly
• Use of Hydrophobic filter minimizes or eliminate concern of filter wetting due to the air moisture content
Types of filters

i. Depth filter - depth filter means, it consists of either multi layer, or single layer of a medium having depth which
captured the contamination of the structure, as oppose to on the surface

ii. Membrane filter – typically traps contaminants larger than the pore size of the membrane on the surface of the
membrane
So, you can say X90 is inversely proportional to K i.e. X90α 1/k, this can be seen in the graph too..
 When, k is maximum, X90 is minimum

The effect of increasing linear air velocity on K and X90

• Decrease of K value at the high velocity is probably due to the disruption of the filter,
allowing the channels to develop and fibers to vibrate and resulting the release of previously
capturing organism.
Drawback - life of the membrane is very less
Hint: so, need to find out X (i.e. how long) and radius (r),
The cross sectional area of the filter (πr2) = volumetric air flow rate/linear air velocity
Problem 3