Sterilization

Decimal reduction time (D)

• Fluid streams can be sterilized through the physical removal of cells
and viruses or the inactivation of living particles by heat, radiation, or
chemicals.
• Decimal reduction time (D) is the time for the number of viable cells
to decrease tenfold.
No=N
Nf=0.1N
D=2.303/kd
Kd is specific death rate
Survival curve-A plot of ln N(t)/N0 versus time is
called a survival curve

N = number of viable spores at any time,

N0 = original number of viable spores.
Sterilization of Liquids
A. Thermal inactivation- preferred for the economic, large-scale
sterilization of equipment and liquids.
B. Radiation- Used for heat-sensitive equipment, media and chemicals.
• Ultraviolet (UV) radiation is effective to sterilize surfaces, but UV cannot
penetrate fluids.
• X-rays cost and safety considerations preclude their use in large-scale
systems.
C. Chemical agents- It must leave no residue that would be toxic to the
desired culture.
• Ethylene oxide is used to sterilize equipment.
• 70% ethanol–water acidified to a pH of 2 with HCl.
• Formaldehyde
• Sodium hypochlorite solution (3%)
• ozone ( affects medium quality)
Thermal sterilization (batch reactor)
• For most large-scale equipment and liquids, thermal sterilization is used.
• Filter sterilization is the only practical alternative for liquids.
• Dependence of the specific death rate on temperature is given by an Arrhenius equation:
Kd=αe-E˳d /RT
where R is the gas constant
T is absolute temperature
E0d is the activation energy for the death of the organism.
• Values for E0d range from about 50 to 150 kcal/g-mol.
• Bacillus stearothermophilus E0d= 70 kcal/g-mol
• E. coli. E0d= 127 kcal/g-mol
• Thermal sterilizations take place at 121℃.
• The time required to heat the fluid to 121℃ and to cool it back to growth temperatures is much longer than
the time of exposure to the desired temperature.
• The elevated temperatures during heat-up and cool-down can be very damaging to vitamins and proteins,
can lead to caramelization reactions for sugars, and can greatly alter medium quality.
A typical temperature-time profile for batch sterilization is
shown in Figure.
Heating time (room temp. to 121℃)
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 time.
Holding time (at 121 ℃)
Once the holding or sterilization temperature is reached,
the temperature is held constant for some time thd.
Cooling time (121 ℃ to room temp.)
Cooling water in the coils or jacket of the fermenter is
then used to reduce the medium temperature to the
required value.
• Holding time required to achieve the desired level of
cell destruction needs to be known.
• Holding times at the highest sterilization temperature
should be kept as short as possible.
Calculation of holding time
• N0 is the number of contaminants present in the raw medium
• During the heating period this number is reduced to N1
• At the end of the holding period the cell number is N2
• The final number after cooling is Nf (Ideally Nf is zero at the end of the
sterilization)
• Absolute sterility would require an infinitely-long sterilization time, it is
theoretically impossible to achieve.
• Normally, the target level of contamination is expressed as a fraction of a
cell, which is related to the probability of contamination (example Nf =10-3)
• If No and Nf are known, we can determine the holding time required to reduce the number of
cells from N1 to N2 by considering the kinetics of cell death.
dN/dt=-kdN
N is number of viable cells
t is time
kd is the specific death constant
dN/dt=-kdN applies to each stage of the batch sterilization cycle: heating, holding and cooling.
• Kd is a strong function of temperature, direct integration of this equation is valid only when the
temperature is constant, i.e. during the holding period.
ln N1/N2 = kd thd

thd= (ln N1/N2 )/ kd

thd is the holding time
N1 is the number of viable cells at the start of holding
N2is the number of viable cells at the end of holding
Kd=Ae-E˳d /RT

dN/dt=-Ae-E˳d /RTN
For heating period

For cooling period

Overall time = heating time+ holding time+ cooling time

𝛁𝒐𝒗𝒆𝒓𝒂𝒍𝒍 = 𝛁𝒉𝒆𝒂𝒕𝒊𝒏𝒈 + 𝛁𝒉𝒐𝒍𝒅𝒊𝒏𝒈 + 𝛁𝒄𝒐𝒐𝒍𝒊𝒏𝒈
𝒍𝒏 𝑵
𝛁𝒐𝒗𝒆𝒓𝒂𝒍𝒍 = 𝟎
=kd.t
𝑵
𝛁 𝒉𝒐𝒍𝒅𝒊𝒏𝒈 =Ae(-E˳d /RT) .t

Richards Rapid Formula

This is an empirical equation given for a particular set of conditions to calculate ∇ ℎ𝑒𝑎𝑡𝑖𝑛𝑔 𝑜𝑟 ∇𝑐𝑜𝑜𝑙𝑖𝑛𝑔
This holds true only when the sterilization is carried out at temperature above 100℃ and holding temp is greater
than 100℃ (ideally 121 ℃)
𝜵 𝒉𝒆𝒂𝒕𝒊𝒏𝒈 𝒐𝒓 𝜵𝒄𝒐𝒐𝒍𝒊𝒏𝒈= C∆𝒕/∆𝑻
t= time
T= temperature
∆𝑻 = 𝟏𝟐𝟏 ℃- 100 ℃ = 21
C=9.6 for Bascillus thermophillus
Generalized temperature-time profiles for the
heating and cooling stages of a batch sterilization
cycle
Thermal sterilization (continuous reactor)
• Continuous sterilization, particularly a high-temperature, short-exposure
time, can achieve complete sterilization with much less damage to the
medium.
• Continuous sterilization is easier to control and reduces downtime in the
fermenters.
• Disadvantages - dilution of the medium with steam injection and foaming.
• The flow pattern inside the pipe is critical, since the fluid residence time
near the wall can be different from that in the center.
• The average flow rate and the length of the sterilizing section should be
designed to ensure high Peclet numbers (above 500), so that the velocity
distribution approaches piston-like flow.
Filter sterilization
• Performed when the medium contains heat-sensitive materials.
• Example- sterilization of medium to support the growth of animal
cells. Microporous filters (pore sizes < 0.2 mm) are typically used.
• The filters should be absolute filters; no pores larger than the nominal
pore size must exist.
• Filter sterilization is not as reliable as steam sterilization.
• Any defect in the membrane can lead to failure.
• Viruses and mycoplasma (small wall-less bacteria) can pass the filter.
• Filtered–sterilized medium is usually quarantined for a period of time.
Comparison of a batch (A) with a continuous sterilization strategy (B) for
the temperature profile of the medium sterilized. The continuous unit allows short-time,
high-temperature sterilization.
Sterilization of Gases
• Adiabatic compression of air can increase the air temperature (typically 150° to
220°C).
• Dry heat is less effective in killing organisms than moist heat.
• To kill spore temperature of 220℃ for 30 s is required.
• Exit air cools rapidly and the pipes connecting the compressor to the fermenter
are difficult to maintain as sterile, an air-filtration step is almost always used.
Filters
• The filtration of gases can be accomplished using either depth filters or surface
filters.
• Depth filters using glass wool were used, but depth filters have been almost
totally replaced with membrane cartridge filters, which are surface filters.
• Possible mechanisms of removal for particles of about 1-mm diameter are direct
interception, electrostatic effects, diffusion (or Brownian motion), and inertial
effects.
dN=f(t,x)
The number of microbes filtered is a function of time and depth of filter.
T=time
X=depth of filter
dN/N=-k.dx
k depends on the linear velocity of the air flow that is impacting the
membrane
V=Q/A
V=linear velocity of air flow
Q= flow rate
A=area of cross section of filter
• Mechanism - As the flowing gas approaches a fiber, the flow streamlines must
curve around the fiber. If a particle had no density, it would follow the
streamlines around the fiber. Only particles whose centers are on streamlines less
than a particle radius away from the fiber could be intercepted. For real particles,
inertial effects mean that particles with mass will have a tendency to maintain a
straight-line trajectory as they approach the fiber. Thus, such particles deviate
from the streamline and crash into the fiber. Both interception and inertial effects
are important in removing bacteria.

• Diffusional effects may be important for virus removal, but bacteria are
sufficiently large that diffusion is relatively unimportant
• The deeper the filter is, the smaller the probability of a particle penetrating the
depth of the filter.
• Depth filters using glass wool can show shrinkage and hardening upon steam
sterilization. In such cases, channeling can occur, and the filter becomes far less
effective.
• Another serious problem with such fibrous filters is wetting. If a filter
wets, an easy path becomes available for a contaminant to penetrate
through it. A wet filter also greatly increases pressure drop. Thus, any
condensation within such a filter must be avoided.
• Surface filters (membrane cartridges) work using another mechanism for
particle removal, a sieving effect.
• Membranes with uniformly small pores prevent the passage of particles
with a radius larger than the pore radius. Such filters can be steam-
sterilized many times. Also, any condensate formed on the non-sterile side
cannot pass into the sterile side.
• With both depth filters and membrane cartridges, pressure drop is critical.
The energy input for compressed air for a commercial-scale process is
significant. Air treatment can account for 25% of total production costs.
• Aerosol in the exit gas from a filter cartridge can be monitored by a
photometer.
• Question
In a 20 m3 fermenter aerobic batch reaction is carried out for 100 hours. Air
is applied to the fermenter broth at a rate of 0.5 vvm. The air contains 200
micro organism m-1 and the air has a linear velocity of 0.15 m/sec and
k=1.535 per cm. calculate the dimension of the filter. The target air should
not contain microbial count of 10-3.

• Question
In a bioreactor you have a moderately temperature sensitive bacteria,
therefore sterilization is done at 70℃ for 5 minutes to reduce the microbial
count to 20% of its initial concentration. Now determine the decimal
reduction time. Estimate exposure time if Nf=0.01% No and then compare
efficiency.
• Question
Efficiency of sterilization is 99%. ∇heating is 8. ∇overall is 22 and ∇cooling is
0. calculate holding time and overall time. Kd=5.37x10-3sec-1

• Question
In a 30 m3 fermenter air is sterilized using filter having 10 cm length. Air is
supplied to the fermenter at a rate of 0.6 vvm. Air contains 150 microbes/m3
and target air achieved has 1/1000 microbe count. Linear air velocity is 0.1
m/s. and filter constant k=1.2 cm-1. How long can we use this filter. Find its
efficiency, ∇factor and radius of the filter.
Question
Initial microbe count is equal to 1011 count/m3 and target count is 10-3.
calculate ∇factor if it takes 20 minute for heating and 15 minutes for
cooling. Also find time of operation.
• A = 9.51 x 1037 min-1
• Ea = 283 kJ/mol
• R= 8.31 JK-1mol-1