Sterilization in Bioreactors

Batch sterilization, Continuous sterilization

Sterilization is a process to kill or inactivate all viable organisms in a culture
medium or in a gas or in the fermentation equipment.
This is however not possible in practice to kill or inactivate all viable
organisms.
Commercial sterilization is therefore aims at reduce risk of contamination to
an acceptable level.
Reasons for Sterilization:
Foreign organisms (contaminants) are undesired for many bio-productions.
Because, production organism must compete with the foreign organisms for
nutrients.

Foreign organisms can produce harmful (or unwanted) products which may
inhibit the growth of the production organisms.
Sterilization Methods:
 Thermal:
preferred for economical large-scale sterilizations of liquids and equipment
 Chemical:
preferred for heat-sensitive equipment
→ ethylene oxide (gas) for equipment
→ 70% ethanol-water (pH=2) for equipment/surfaces
→ 3% sodium hypochlorite for equipment
 Irradiation:
→ ultraviolet for surfaces
→ X-rays for liquids (costly/safety)
 Filtration:
preferred for heat-sensitive material
Thermal Sterilization: Dry air/steam or moist (wet) steam can be used as the
heat agent. Moist (wet) steam is more effective than dry air/steam.

Thermal sterilization Thermal sterilization

using dry air using moist steam
 Pasteurization (below 100 oC)
 Direct flaming Destroys pathogens without altering the flavor of the food.
Classic method: 63°C; 30 min
High Temperature/Short Time (HTST) : 71.7 °C; 15-20 sec
 Incineration Ultra High Temperature (UHT) : 135 °C; 1 sec
 Boiling (at 100 °C)
killing most vegetative forms microorganisms
 Hot air oven
Requires 10 min or longer time
170 °C for 1 hour Hepatitis virus can survive for 30 min & endospores for 20
h
140 °C for 3 hours Autoclaving (above 100 °C)
killing both vegetative organisms and endospores
121-132 °C; 15 min or longer
 Batch Sterilization
• The target of sterilization is to destroy bacterial spores (bacterial
spores are difficult to destroy).
• Liquid medium is most commonly sterilized in batch in the vessel.
• Types of batch sterilization: Steam heating (by introducing steam into
the coils or jacket of the vessel), direct steam sparging (steam is
bubbled directly into the medium) and electrical heating.
Temperature-time profile for batch sterilization
Depending on the rate of heat transfer
from the steam or electrical element,
raising the temperature of the medium in
large bioreactors can take a significant
time period.
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
bioreactor is then used to reduce the
medium temperature to the required
value.
Usually, holding periods take minutes whereas heating and cooling of large liquid volumes take hours.
Furthermore, longer holding times are required to treat solid-phase substrates and media containing particles.
Batch Sterilization Problems
• Even if one organism is left alive, the whole bioreactor may be
contaminated.
• As well as destroying contaminant organisms, heat sterilization also
destroys nutrients (vitamins, proteins and sugars) in the medium. To
minimize this loss, holding times at the highest sterilization temperature
should be kept as short as possible.
• In sterilization, undesirable reactions may occur between the culture
components. (Ex: between carbohydrates and amine groups,). So, these
substances must be sterilized separately. Otherwise, inhibited growth
products may be formed.
 Reduction in number of viable cells during batch sterilization
If NO and Nf are known, we can determine the
holding time required to reduce the number of
Cell death occurs at cells from N1 to N2 by considering the kinetics of
all times during cell death.
batch sterilization

where thd is the holding time (h), N1 is the number

of viable cells at the start of holding, and N2 is the
number of viable cells at the end of holding, kd is
evaluated as a function of temperature using the
The number of contaminants present in Arrhenius equation:
the raw medium is presented by N0. where kd is the specific death constant (h-1), A is the
During heating period this number is Arrhenius constant or frequency factor (h-1), Ed is the
reduced to N1. At the end of the holding activation energy for the thermal cell death (J/gmol), R is
period, the cell number is N2; after the ideal gas constant (J K-1 gmol-1)and T is absolute
cooling, the final cell number is Nf. temperature (K).
Cells are so resistant to heat when the value of kd is small
Slope shows that cells are affected by heat
 Design criterion for sterilization (▼)
• kd is not constant during the batch sterilization
due to the temperature change in heating and
cooling steps. Therefore, the design criterion is
defined.
• Deindoerfer (1959) used the term «In (N1/N2)» as
a design criterion for sterilization, which has been Due to the negative sign

variously called Del factor and represented by the

term of ▼
• The design criterion was expressed as

• Thus, Del factor gives an idea about fractional reduction in viable organism in a particular
time.
• For a typical sterilization process, the contribution of sterilization steps to the cell
destruction:

20% of the cells die at the heating step, 75% of the cells die in holding step, 5% of the cells die at the cooling step
Temperature profile during the heating period of batch sterilization for steam sparging

Cp= specific heat capacity of medium

h= specific enthalpy difference between steam and raw medium
Mm= initial mass of medium
mass flow rate of steam
T= temperature
To= initial temperature of medium
t= time
Temperature profile during the heating period of batch sterilization for electrical heating

Cp= specific heat capacity of medium

Q= rate of heat transfer
Mm= initial mass of medium
T= temperature
To= initial temperature of medium
t= time
Temperature profile during the heating period of batch sterilization for steam heating

Cp= specific heat capacity of medium

Mm= initial mass of medium
T= temperature
U= overall heat-transfer coefficient
A= surface area for heat transfer
To= initial temperature of medium
Ts= steam temperature
Temperature profile during the cooling period of batch sterilization

Cp= specific heat capacity of medium

Cpw= specific heat capacity of cooling water
Mm= initial mass of medium
mass flow rate of cooling water
T= temperature
U= overall heat-transfer coefficient
A= surface area for heat transfer
To= initial temperature of medium
Tci= inlet temperature of cooling water
Continuous Sterilization
Continuous sterilization eliminates the disadvantages of batch
sterilization.
It is a high temperature and short time (HSTS) process. It is the flash
heating method.
Advantages:
Steam utilization capacity increases.
Product yield increases, because the nutritional values of the components
are not lost much.
Overall production yield increases due to the shorter sterilization time.
Process control is easy.
High-temperature and short time process (HTST)

High-temperature and short-exposure-time process (HTST) can

significantly reduce the damage to the medium ingredients while
achieving high levels of cell destruction.
Other advantages include improved steam economy and more
reliable scale-up. The amount of steam needed for continuous
sterilization is 20-25%.
 The time required is also significantly reduced because heating and
cooling are virtually fast.
Continuous steam injection with flash cooling
When steam is introduced into the raw medium,
condensation may occur and the medium concentration
may be diluted. Therefore, the culture medium should
be prepared more intensely when using this method.
• Raw medium entering the system is first pre-heated in
a heat exchanger.
• Holding section: Steam is then injected directly into
the medium as it flows through a pipe; the liquid
temperature rises to the desired sterilization
temperature. The time of exposure to this
temperature depends on the length of pipe in the
holding section of the sterilizer.
• After sterilization, flash cooling is applied under
vacuum. Further cooling takes place in the heat
exchanger where residual heat is used to pre-heat
incoming medium.
Heat transfer using heat exchangers

• It is based on heat exchange between steam and

medium.
• Raw medium is pre-heated in the heat exchanger
then brought to the sterilization temperature by
steam in the other heat exchanger.
• The sterilization temperature is maintained in the
holding section before being reduced to the
fermentation temperature by heat exchange with
incoming medium.
Dimensionless Groups For Sterilization

 Peclet number

where Pe is the Peclet number, u is the average linear fluid velocity, L is

the pipe length and is the axial-dispersion coefficient. For perfect
plug flow, is zero and in practice, Peclet numbers between 3 and 600
are typical, in practise.
• Damköhler number
where kd is the specific death constant, L is the length of the holding
pipe and u is the average linear liquid velocity.
Filter Sterilization of Liquids
• If culture media containing heat-sensitive components such as enzymes and
serum are easily destroyed by heat, they must be sterilized by filtration.
• Membranes used for filter sterilization of liquids are made of cellulose esters
or other polymers and have pores between 0.2 and 0.45 µm in diameter.
• The membranes themselves must be sterilized before use, usually by steam.
• As medium is passed through the filter, bacteria and other particles with
dimensions greater than the pore size can not pass out the membrane and
they collect on the surface of the membrane.
• Liquid filtration is generally not as effective or reliable as heat sterilization.
Viruses and mycoplasma are able to pass through the membrane filters.
 Sterilization of Air
• Filtration is the most common method for sterilizing air in
large-scale bioprocesses.
• Depth filters consisting of compacted beds or pads of fibrous
material such as glass wool have been used widely in the
fermentation industry.
• Increasingly, depth filters are being replaced for industrial
applications by membrane cartridge filters.
• Air vent filter is used for laboratory scale production.

Membrane cartridge filter

 STERILIZATION FORMULAS

u = The linear velocity in the holding section of the sterilizer (m/h)

F = The volumetric flow rate (m3 h-1)
N1 = The number of cells entering the sterilizer
N2 = The number of cells remaining at the end of the sterilization treatment
thd = Holding time (h)
kd =The specific death constant (h-1)
Da = Dimensionless Damköhler number
L = The length of the holding section (m)
A =Arrhenius constant (h-1)
Ed = Activation energy (J/gmol)
R = Ideal gas constant (J K -1 gmol -1)
T = Temperature (K)
 Q1

A steam sterilizer is used to sterilize liquid medium for fermentation. The initial
concentration of contaminating organisms is 108 per litre. For design purposes, the final
acceptable level of contamination is usually taken to be 10-3 cells per m3.
For how long should 1 m3 medium be treated if the temperature is:
(a) 80 °C
(b) 121°C
(c) 140°C
To be safe, assume that the contaminants present are spores of Bacillus
stearothermophilus, one of the most heat-resistant microorganisms known. For these
spores the activation energy for thermal death is 283 kJ gmol-1 and the Arrhenius
constant is 1036.2 s-1. The ideal gas constant is 8.3144x10-3 kJ K-1 gmol-1.
A1
 A1

11
10
ln ⁡( − 3 )=𝑘𝑑 𝑡 h𝑑
10

14
ln ⁡(10 )=𝑘 𝑑 𝑡 h𝑑
SAME
32.24
3 2.24=𝑘𝑑 𝑡 h𝑑 𝑡 h𝑑=
𝑘𝑑
A1
 Q2

Medium at a flow rate of 2 m3 h-1 is to be sterilized by heat

exchange with steam in a continuous sterilizer. The
activation energy and Arrhenius constant for thermal
destruction of the contaminants are 283 kJ gmol-1 and 5.7
x 1039 h-1, respectively. The sterilizer pipe has an inner
diameter of 0.1 m; the length of the holding section is 24
m. What sterilizing temperature (as °C) is required?
Assume that: Damköhler number (Da) is 42. The ideal gas
constant is 8.3144 J K -1gmol -1.
A2
 Q3
A 15-m3 reactor is operated with dilution rate 0.1 h -1. A
continuous sterilizer with steam injection with flash cooling
delivers sterilized medium to the fermenter. Medium in the
holding section of the sterilizer is maintained at 130 °C. The
concentration of contaminants in the raw medium is 105
cells/ml; an acceptable contamination risk is one organism
every 3 months. The acceptable number of cells remaining at
the end of the sterilization treatment is 1 cell. The Arrhenius
constant and activation energy for thermal death are estimated
to be 7.5 x 1039 h-1 and 288.5 kJ gmol-1, respectively. The
sterilizer pipe inner diameter is 12 cm. T he ideal gas constant
is 8.3144 J K-1gmol -1. Assuming perfect plug flow, determine
the linear velocity in the holding section of the sterilizer as m/h
and the sterilization time as h.
 A3

𝒄𝒆𝒍𝒍𝒔 cells
𝒎𝒍