3

Sterilization Techniques used

in Fermentation Processes
Shivani Sharma, Arindam Kuila and Vinay Sharma*

Department of Bioscience & Biotechnology, Banasthali University, Rajasthan, India

Abstract
Fermentation is a microbial process that must be perfectly sterilised. Apart from
the normal sterilization sometimes this process is being operated on a large scale
that employs large fermenting vessels that need to be sterilised. Media that are
being used also requires sterilization. Major problem in the process of fermenta-
tion is contamination, in this chapter we will be reviewing some of the processes
of sterilization that includes sterilization of the reactor vessel, media, air, and pipes
through which the media passes.

Keywords: Fermentation, sterilization, microbial death

3.1 Introduction
Fermentation comes from a Latin word ‘fermentare’ that means to boil,
that can be seen as a boiling appearance of the most common fermenting
microorganism Yeast on the fruits, grains and other materials that undergo
fermentation. The carbon dioxide produced during the anaerobic process
that degrades the sugars in the fruits and grains forming bubbles that give
boiling appearance. Fermentation is done by the inoculation of desirable
organism or a group of organisms in the suitable media. But if this fer-
menting media comes in contact with any foreign microorganism it may
lead to a loss in productivity of the actual fermenting microorganism due
to a couple of reasons that are listed below.

*Corresponding author: vinaysharma30@yahoo.co.uk

Arindam Kuila and Vinay Sharma (eds.) Principles and Applications of Fermentation Technology,
(45–52) © 2018 Scrivener Publishing LLC

45
46 Principles and Applications of Fermentation Technology

1. The foreign microorganism can outgrow the actual ferment-

ing microorganism
2. The final product can be contaminated
3. The extraction of final product can become difficult due to
the formation of some complex compound by the help of
foreign microorganisms.

This can be avoided by the following methods.

1. Inoculation in a pure culture

2. Sterilization of the media used
3. Sterilizing the equipments and containers used
4. Aseptic conditions should be maintained during the whole
process of fermentation.
5. In order to obtain proper fermentation first the media used
is to be sterilised. Sterilization of media can be done by use
of radiation, chemical treatment, filtration, ultrasonic treat-
ment or heat. The sterilizing treatment given is referred to
as sterilant. Heating is the most commonly used form of
sterilization as it kills most of the endospores present in the
media. Little amount of heat is given in the form of com-
mercial sterilization that kills most of the microorganisms
present in the media. Mostly sterilization with the help of
steam is recommended for fermentation and all the other
work best for animal or plant cultures.

3.2 Rate of Microbial Death

Death at constant rate occurs when the media is contaminated by some
microorganism and heat or any other sterilizing treatment is being given.
For example, when heat treatment is given for one minute then 90% of the
population dies. To kill the remaining population again the same treatment
is given then again 90% of the population dies. To kill the whole population
we have to repeat the treatment and for this process if a graph is plotted
then we get a constant curve for the death rate of microorganism.
Some factors listed below are responsible for the way a sterilizing treat-
ment works.

Total microbes: if the number of microbes present is large then

too much time would be required to kill them.
 Sterilization Techniques used in Fermentation Processes 47

Environment required: warm environment is best suited for

most of the sterilizing actions.
Nature of the media: if the media is rich in proteins and fats then
the microbes will not be easily killed; mostly heating treat-
ment helps in these cases.
Duration of treatment: different sterilizing treatments need dif-
ferent time of exposure as chemical sterilizing treatments
require more time compared to normal heating methods.
Characteristics of the contaminant present: the type of microor-
ganism present in the contaminant also affects the choice of
the treatment that would require to eradicate it.

3.3 How do Sterilants Work?

Most of the sterilizing treatments used are aimed at killing the contami-
nants present in the media used. There are different ways in which the con-
taminants can be killed.

Changing the permeability of cell membrane: The plasma mem-

brane present inside the cell membrane of the contaminant
is the target of sterilizing agents, as it is responsible for the
exchange of nutrients in the cell and removal of wastes.
The sterilizing agents used, cause disturbance to the plasma
membrane that leads to improper exchange of nutrients
and leakage of the cell components leading to death of the
contaminant.
Destroying the proteins and nucleic acids present: The microbes
that act as contaminants are mostly bacteria. Bacteria are
rich source of enzymes and enzymes are proteins that are
very fragile to heat treatment as they easily denature. DNA
and RNA that carries the genetic information of the microbe
can also be easily destroyed by any kind of sterilizing treat-
ment, like heating, radiation or chemical treatment.

3.4 Types of Sterilization

During the early Stone Age, humans used several methods of sterilization
like salting and drying. Heating is one of the most favourable methods of
sterilization since early days. But time introduced some microbes that are
48 Principles and Applications of Fermentation Technology

heat resistant, so various other methods are required. Heat, pressure and
radiation all play their role in different types of physical sterilization.

3.4.1 Heat
Heat is preferred because it kills the microorganisms by denaturing the
enzymes present in them, hence changing the three dimensional structure
of proteins which therefore inactivates them. Three factors that decides the
action of heat sterilization are as follows.

Thermal death point: minimum temperature required to kill

microbes present in a suspension in 10 minute.
Thermal death time: minimum time required to kill microbes
present in a suspension at a given temperature.
Decimal reduction time: time, in minutes that is required to kill
microbes at a given temperature.

Heat treatment can be given using moist heat or dry heat sterilization.
Dry heat sterilization involves killing of the oxidation effects by flaming
or incineration. Moist heat sterilization works by coagulating the proteins
present in the microbes thereby killing them. Autoclaving is the most
common method of moist heat sterilization in which steam is used to kill
microbes at high temperature and under appropriate pressure. This helps
in sterilizing glassware, culture media, equipments, solutions etc.

3.4.2 Pressure
At high pressure molecular structure of proteins and carbohydrates are
altered leading to inactivation of cells.

3.4.3 Radiation
Depending on the intensity, wave-length and duration radiation has vari-
ous effects on the microbes. Two types of radiations kill microbes, they are
Nonionizing and Ionizing radiations.

Nonionizing radiation: They have wavelength greater than

1 nm. Ultraviolet light is a form of nonionizing radiation, it
kills the microbes by degrading their DNA by making thy-
mine dimers. These dimmers prevent the replication of DNA
during cell reproduction. UV wavelength of 260 nm is very
 Sterilization Techniques used in Fermentation Processes 49

effective in killing the microbes. These radiations are effec-

tive in sterilizing glassware, air, culture media etc.
Ionizing radiation: They have wavelength less than 1 nm, like
the X rays, gamma rays, electron beams of high energy. These
radiations work by ionising the water and forming highly
reactive hydroxyl radicals. These radicals kill microbes by
causing some damage to DNA or RNA by a series of reactions.

3.4.4 Filtration
It helps in removal of microbes by making any liquid or gas pass through
a membrane with pores. Pressure is applied to make the liquid or gas pass
through the filter kept on a container in which vacuum is being created.

3.4.5 Steam Sterilization

Sterilization by steam can be done by the use of normal steam created by
preventing evaporation of the medium or with the help of steam under
pressure used in autoclaves. Koch or Arnold steamer is used that contains a
copper cabinet with lagged walls and conical lid that helps in the drainage of
steam generated and a perforated tray is placed above water level that helps
in proper distribution of steam. Temperature 100 °C for 20 minutes for three
days is required and the process is known as intermittent sterilization. This
method efficiently kills bacteria but is inefficient in killing anaerobic spores.
Another type of steamer used is autoclave that generates steam under
pressure where water boils when its pressure is equal to the atmospheric
pressure in the vessel and with the increase in pressure, temperature of
water increases.

3.5 Sterilization of the Culture Media

This can be done by heating treatment, that is, autoclaving or by the fol-
lowing two ways.

3.5.1 Batch Sterilization

This can be done in a fermentation vessel or separated cooker. When the
fermenters are being cleaned and prepared for fermentation the media
used can be sterilized in cookers. One cooker can be used for several fer-
menters. Concentrated form of media can be used for sterilization and
that can be diluted when placed within the fermenter, this helps in saving
50 Principles and Applications of Fermentation Technology

time. As one cooker is mostly used in this type of sterilization so a complex

pipeline network is required that connects all the fermenters delivering the
sterilized media. This type of sterilization makes sure that there is minimal
loss of nutrients as it is operated at 121 °C that is almost feasible for all
nutrients present in the media.

3.5.2 Continuous Sterilization

In this type of sterilization the medium is heated to a particular tempera-
ture, it is kept at the same temperature for some time and then cooled. The
time at which media is kept at a certain temperature is known as holding
time. A continuous heat exchanger is used to heat the media and then insu-
lated serpentine coiled tube is used to keep it at that temperature for the
holding period. Sequential heat exchangers are used to cool the media to
suitable fermentation temperature. Two types of continuous sterilizers are
used, one that uses direct heat by steam injection and the other uses indirect
heat. Small increments of media are done continuously at certain time inter-
vals and the heating and cooling times are properly managed. Spiral heat
exchangers are used in cooling the heated media with the help of unsterile
media that come for sterilization. This unsterile media gets heat from the
sterile hot media thus preserving the heat before reaching the sterilizer.
Continuous steam injectors are used to directly inject the heat into the
media, but it has certain pros and cons [2]: this is very affordable and man-
ageable procedure with very short heating time but it produces foam on
heating that can cause contamination. Sometimes this steam injector is
combined with flash cooling, in which a vacuum chamber is made hav-
ing an expansion valve through which the media is passed, this causes
instant cooling of the media. Sometimes a combination of direct and indi-
rect heat exchangers is being used mainly for starch rich media [3]. Spiral
heat exchangers are the best choice for continuous sterilization. A plant
of interconnected spiral heat exchangers and holding tube is made. Hot
water is used to sterilize this plant in a closed circuit before unsterilized
media comes in. The fermenter and holding tube are steam sterilized. Heat
is conserved in this procedure by cooling the sterilized heated media with
the help of cool unsterilized media.

3.6 Sterilization of the Additives

During the course of fermentation some additives are being introduced
in order to increase the efficiency of the process. Continuous and batch
 Sterilization Techniques used in Fermentation Processes 51

sterilization methods are used for the sterilization of additives but the
process depends on the biochemical nature and the volume to be intro-
duced. Continuous sterilization is preferred when large volume of addi-
tive are used [1]. Batch sterilization uses steam injection into the additive
liquid used.

3.7 Sterilization of the Fermenter Vessel

The fermenter vessel is to be sterilized before the fermentation begins as
the media used is sterilized in separate cookers. The coils or jackets of the
fermentation vessel are heated with the help of steam that is introduced into
it from various entries and one valve is opened through which the steam
goes out of the vessel very slowly. Steam is introduces at 15 psi pressure for
20–25 minutes. Sterile air is removed out of the vessel by sparing and a posi-
tive pressure is maintained that prevents formation of vacuum in the vessel.

3.8 Filter Sterilization

Suspended fluids that are present in the media can be sterilized with the
help of filter sterilization by the following methods.

3.8.1 Diffusion
Brownian motion is introduced for the separation of very small particles
present in the fluid by collision with each other. Small particles deviate
from the normal flow of fluid and get caught in the filter fibres. This pro-
cess is more effective in the filtration of gases.

3.8.2 Inertial Impaction

The suspended particles present in the fluid have some kind of momentum
based on which they are separated with the help of this method. A route of
less resistance is created in which the fluid flows through the filter and the
suspended particles, due to their momentum flow in a straight line and get
trapped in the filter.

3.8.3 Electrostatic Attraction

The filtration membrane has charge that attracts the opposite charged sus-
pended particles.
52 Principles and Applications of Fermentation Technology

3.8.4 Interception
Interwoven fibres make the filter with openings of different sizes. Particles
that are larger than the pore size are excluded and sometime smaller par-
ticles are also unable to cross the filter membrane as their passage gets
blocked by other particles and they are removed.

3.9 Sterilization of Air

Anaerobic fermentation needs continuous addition of sterile air that can
either be sterilized by heat or filtration. Filters with fixed pores are mostly
used for the sterilization of air. Hygroscopic materials like PTFE are used in
theses filters for this purpose. Other filters used to purify air are as follows.

Depth filters: they consist of glass wool in which the microbes

present in the air gets trapped. These filters work by inertia,
gravitation, diffusion, electrostatic attraction etc. These glass
wool filters can be easily reused after sterilization. But after
sometime due to the action of steam theses glass wool filters
get damaged, so now glass fibre cartridges are used.
Membrane cartridge filters: these filters are made of nylon, cel-
lulose or polysulfone and are pleated. They can be easily used
and operated.

References
1. Anustrup K., Andresen O., Flach E. A., Nielsen T. A., Production of microbial
enzymes. Microb. Technol., 1, 282–309, 1979.
2. Banks G. T., Scale up fermentation process. Top. Enzyme Ferment. Biotechnol.,
3, 170–266, 1979.
3. Svensson R., Continuous media sterilization in biotechnological fermentation.
Dechema Monogr., 113, 225–237, 1988.