See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/358265334

Media and Air Sterilization Required for Microbial Fermentation 1*

Book · February 2022

CITATIONS READS
0 3,633

4 authors, including:

Constance Chinyere Ezemba Arinze Steven Ezemba

ChyChy Gilgal Laboratories and Consultancy @Chukwuemeka Odumegwu Ojukwu U… Nnamdi Azikiwe University, Awka
133 PUBLICATIONS 228 CITATIONS 42 PUBLICATIONS 26 CITATIONS

SEE PROFILE SEE PROFILE

All content following this page was uploaded by Constance Chinyere Ezemba on 01 February 2022.

The user has requested enhancement of the downloaded file.

Media and Air Sterilization Required for Microbial Fermentation
1*
Ezemba Chinyere Constance: 2Obi Chisom Perpetua and 3Ezemba Arinze Steve

AFFLIATED INSTITUTION:
1
Department of Microbiology, Chukwuemeka Odumegwu Ojukwu University (COOU) P.M.B 02, Uli, Anambra
State, Nigeria.
2
Department of Science Laboratory and Technology,Federal Polytechnic Oko.Anambra State.
3
Department of Applied Microbiology and Brewing, Nnamdi Azikiwe University Awka, Anambra State, Nigeria.
E-MAIL:* constancechinyere790@gmail.com

Abstract
Sterilization of media and air for microbial fermentation entails the removal, killing, or destruction of all forms
of life, including bacteria, viruses, spores, fungi, and other microorganisms. This is critical in order to ensure
the sterility of the media containing the required nutrition, the sterility of the incoming and outgoing air, as
well as the sterility of the bioreactors, and the avoidance of contamination during the process. This can be
accomplished through batch sterilization, in which the medium is sterilized at 121°C in batch volumes in the
bioreactors, either directly or indirectly, and continuous sterilization, in which the sterilization is carried out
for a short period of time (30 to 120 seconds) at 140°C. To avoid contamination of the fermentation process,
proper sterilization necessitates continual and vigorous aeration of sterile air in the bioreactor. Different
means, such as chemical, radiation, physical, and heat, can be used to accomplish this. Thus, the filtration
method of air sterilization is the most widely employed in industries for fermentation purposes, in which air is
permitted to pass through filters rather than electrically heated devices, which is relatively expensive. While
the heat method of media sterilization is extensively utilized, this is due to the quality and quantity of
contamination. The components of the media, as well as its pH and suspended particle sizes, are all important
elements in the sterilization's success. As a result, chemical and radiation sterilizing procedures are rarely
used.
Key Words: Microorganisms, Sterilization, Air and Media.

1.0 Introduction
Sterilization is the complete elimination or annihilation of all living microorganisms in or on an object being
sterilized (vegetative cells 60oC 5-10 minutes and spores 80oC 15-20 minutes). To achieve a successful
fermentation, you must assure the following:
-Sterility of the nutrients-containing media,
-Sterility of the incoming and exiting air
-Sterility of the bioreactor.
- Prevention of contamination during fermentation (William, 2008).

Almost all fermentation methods necessitate cost-effective approaches in order to produce a culture that is
free of contamination throughout the whole process, from start to finish. A bioreactor can be sterilized by
eliminating all bacteria using a noxious substance such as heat, radiation, or chemical, or by physically
removing the living organism using filtration.(Stanbury, 2017).

Culture medium components such as water and containers contribute to microorganism contamination,
whether by spores or vegetative cells, so the media must be clear of contamination before being used for
fermentation. As a result, for a successful fermentation, the air must be sterile and free of all microorganisms
and suspended particles (Bansod, 2021). As a result, the amount of suspended particles and microorganisms

1
in the ambient outside air varies greatly. As a result, whatever method of media and air sterilization is chosen,
the procedure must be validated for each type of product or material, both in terms of sterility assurance and
to guarantee that no detrimental changes have occurred within the product. A non-sterile or degraded
fermented product could result if a specific, validated method is not followed properly (Stanbury, 2017). A
typical validation program for steam or dry-heat sterilization entails correlating temperature measurements
taken with sensory devices to demonstrate heat penetration and heat distribution with the destruction of
biological indicators, such as preparations of specific microorganisms known to be resistant to the sterilization
process (Wells-Bennik, 2019). Biological markers are also used to confirm various sterilization procedures and
to monitor specific cycles on a regular basis. Revalidation should be done on a regular basis.

1.1 The Fermentation Media

Fermentation medium is a type of media used in industries to grow microorganisms. It must have all of the
necessary ingredients in a usable form for the production of cellular compounds and metabolic products. The
following are some examples:
1. Primary Metabolites (ethanol and citric acid), which are growth-related products; product synthesis is
directly dependent on microbe development, thus the medium should be conducive to excellent
growth.
2. Secondary Metabolites (antibiotics, alkaloids, Gibberellins) that are not directly related to growth, as
well as substrate measurements for product formation must also be considered.

The media used for the growth of microorganisms in industrial fermentation must contain all the elements in
a suitable form for the synthesis of cellular substances as well as the metabolic products. While designing a
medium, several factors must be taken into consideration. The most important among them is the ultimate
product desired in the fermentation.

All of the components necessary for the creation of cellular compounds as well as metabolic products must be
present in the media used to cultivate microorganisms in industrial fermentation. Several aspects must be
taken into account when creating a media (Sergio, 2008).The most essential of these is the fermentation's
desired end product.
Because the formation of growth-linked products (primary metabolites such as ethanol and citric acid) is
directly dependent on the growth of the organisms, the medium should be conducive to healthy growth. On
the other hand, the substrate requirements for producing the product must be considered for products that
are not directly linked to growth (secondary metabolites such as antibiotics, alkaloids, and gibberellins).
(Sikander, 2018). For cultivating microorganisms in the laboratory, pure specified substances can be
employed. However, for economic reasons, indeterminate and complex substrates are routinely utilized in
industrial fermentations. Cheaper substrates are advantageous since they reduce the cost of fermented goods
manufacture.(Rajana, 2020). Agricultural waste and other industry leftovers are often favored, however their
composition varies greatly. Because of seasonal fluctuations, the cost of raw materials used in fermentation is
heavily influenced by their cost at the moment. For a good product formation, the medium chosen is
extremely important. Microorganisms in general use a luxury metabolism for industrial fermentation. With a
plentiful supply of carbon and nitrogen sources, as well as the necessary growth ingredients, good output
yields can be predicted (Obi, 2016). As a result, the eventual products desired in the fermentation are the
most important aspects to consider while constructing a medium.

2
The design of culture media is divided into three steps, as shown below.
STEP 1: Determine the nutrients required for cell growth and the generation of the target metabolites by
combining all non nutritive elements (anti-foaming agents, surface active agents, buffers).
Selective media may be required to inhibit the growth of undesirable microorganisms while promoting the
growth of desired microorganisms.
STEP 2: Determine the origins of such components, based on the variables listed below, before determining
which media to use for a specific microorganism.

1. Materials cost—waste products from industries, such as molasses from sugar cane and corn steep
liquor from starch, may be cost-effective.
2. Ready Availability; If seasonal or imported, production may be halted, and storage is costly (e.g.
cassava, yam).
3. Transportation costs—the closer the source, the better, assuming all other conditions are met.
4. Ease of disposal of trash generated by raw materials; Some trash is often found to be beneficial as
raw materials in other businesses, such as leftover grains used as animal feed by breweries. It's a time-
consuming and expensive process with a lot of government regulation.
5. Consistency in raw material quality; its composition must be reasonably constant in order to
maintain uniformity of quality in the finished product, as well as ease of standardization or consistency
to the customer's satisfaction and expectations. As a result, before each batch of raw materials is
utilized, it must be chemically evaluated to determine how much of each nutrient must be supplied.
6. Presence of important precursors for vitamin D synthesis; such as cobalt in colbalt media. Usually,
phenyl component in corn steeps for penicillin G. Amino acids are frequently employed to boost
secondary metabolite production, either directly, indirectly or both, such as by increasing the quantity
of a limiting metabolite or Either By inducing a biosynthetic enzyme.
7. Satisfaction of their growth and production requirements; high levels of glucose and phosphate
inhibit the onset of two phases of growth in industrial batch cultivation, such as the Idiophase (phase
of production) and Trophophase (phase of growth), in the production of a number of secondary
metabolites of industrial importance, which will negatively affect production, and other factors such as
-stability,
-purity,
-sources of other media components,
-easy transportation,
-handling
- storage.
STEP 3: Determine the exact concentration of each component. Maybe that's the case which is required for
product production rather than biomass synthesis, which can be gathered or done by
a. Analysis of cell biomass composition
b. Stoichiometry analysis (cell growth, product creation, and empirical approaches) is the study and
determination of quantitative relationships between reactants and products in chemical reactions
(Marcel, 2010).

1.2. Synthetic or Crude Medium can be Utilized in Fermentation Operations:

Synthetic media: can be defined as media that contains all of the required ingredients in their purest form in
the desired proportion.

3
Crude media: They are non-synthetic media made from naturally occurring sources.

-Synthetic media in fermentation is not feasible due to cost; however pure specified defined substances can
be utilized in the laboratory for cultivating microorganisms. So cheaper substrates, such as waste raw
materials that are inexpensive, widely available, and chemically consistent, are favorable since they reduce
the fermented product's production cost, Using local replacements whenever possible saves money on
transportation and even creates jobs for the local community (Sergio, 2012). Wastes from agriculture and by
products of other industries are generally preferred, although they are highly variable in composition. Raw
materials used in fermentation largely depend on the cost at a particular time, since there are seasonal
variations.

In an industry where microorganisms in general use a luxury metabolism, the medium chosen is important for
successful product production. In practice, crude medium with the inclusion of essential synthetic ingredients
is excellent for high product production and a plentiful supply of carbon, nitrogen sources, and growth factors
for industrial fermentation(Sergio, 2012).

The most frequently used substrate for industrial fermentation with special reference to the supply of carbon
and nitrogen sources and growth factors are as stated below

1.3. Substrates Used As Carbon Sources:

Carbohydrates are the most common energy source in the fermentation sector. For economic reasons,
refined and pure carbohydrates such as glucose or sucrose are rarely used (Kiro, 2010).

-Molasses:
Molasses is a sugar industry waste that is one of the cheapest sources of carbohydrates. Rice in sweet
nitrogenous compounds, vitamins, and trace elements are often utilized. Sugar cane molasses (sucrose
around 48 percent) and sugar beet molasses (sucrose around 33 percent) are commonly used. Molasses also
contains nitrogenous compounds, vitamins, and trace elements in addition to sugar. The composition of
molasses varies, and it is largely determined by climatic conditions and the manufacturing process. The
byproduct of maize glucose synthesis, hydrol molasses, is also employed as a fermentation substrate (Bansod,
2021).

-Rice:
Rice in sugary nitrogenous substances, vitamins and trace elements are commonly used.

-Corn steep liquor:

Corn Steep Liquor is a by-product of maize starch production.

-Malt extract:
Malt extract is a water-based extract of malted barley that comprises roughly 80% carbohydrates (glucose,
fructose, sucrose, and maltose). Proteins, peptides, amino acids, purines, and pyrimidines account for about
4.5 percent of nitrogenous compound.

-Cellulose, dextrin, and starch:

Microorganisms can metabolize polysaccharides such as, cellulose dextrin, and starch. They're widely utilized
in the manufacturing of alcohol in the industrial sector. The use of cellulose for alcohol production is
intensively explored due to its vast availability and low cost (Singh, 2019).
4
-Whey
Whey is a byproduct of the dairy industry that is generated all over the world. Humans and animals consume
the most of it. Whey is a good source of carbon for ethanol, single-cell protein, vitamin B12, lactic acid, and
gibberellic acid production. Whey storage is a barrier to its broad use in the fermentation sector (Bansod,
2021).

-Methanol and ethanol

Some microbes have the ability to use methanol and/or ethanol as a carbon source. Methanol is the cheapest
fermentation substrate. Only a few bacteria and yeasts, however, can use it (Matuszewska, 2016).Methanol is
a common ingredient in single-cell protein synthesis. Ethanol is quite costly. It is, nevertheless, currently
employed in the manufacturing of acetic acid.

-Growth Factors

They are microbes that are not capable of synthesizing nutrients such as vitamins, minerals (phosphate,
sulfate) thus are being supplemented.

1.3. Substrates Used As Nitrogen Sources:

The fermentation bacteria can get nitrogen from both inorganic and organic sources (Costa et al,2002).
 Sources of inorganic nitrogen include:
Inorganic nitrogen sources such as ammonium salts and free ammonia are very inexpensive in industrialized
countries. However, not all bacteria can utilize them, therefore their application is limited or restricted.
 Sources of organic nitrogen include:
Urea is a somewhat good nitrogen source. Other, less expensive organic nitrogen sources are preferred.

-Corn-steep liquor:
This is produced when corn is used to make starch. Corn steep liquor is high in nitrogen (approximately 4%),
and microbes use it quite efficiently. It's high in a variety of amino acids (alanine, valine, methionine, arginine,
threonine, glutamate).

-Yeast extracts:
They are high in amino acids, peptides, and vitamins and contain roughly 8% nitrogen. Glucose produced
during yeast extraction from glycogen and trehalose is a useful carbon source (Singh, 2019). Autolysis (at 50-
55°C) or plasmolysis is used to obtain yeast extracts from baker's yeast (high concentration of NaCI). Many
industrially important microorganisms can be found in yeast extracts.

-Soy meal;
Soy meal is the leftover residue after extracting the soy bean oil from the soy bean seeds. It has a high protein
level (about 50%) as well as high carbohydrate content (around 30%). Antibiotics are frequently made from
soy meal.

5
-Peptones:
Protein hydrolysates are referred to as peptones collectively, and they are a good source of nutrients for
many bacteria (Bansod, 2021). Meat, soy meal, peanut seeds, cotton seeds, and sunflower seeds are all
sources of peptones.
Casein, gelatin, and keratin are all proteins that can be hydrolyzed to produce peptones. Peptones generated
from animal sources have a higher nitrogen level, whereas those obtained from plants have higher
carbohydrate content (Costa, 2002). Peptones are found in soy meal, peanut seeds, cotton, and sunflower
seeds, among other plants. While the animal sources include, meat Casein, gelatin, and keratin are hydrolyzed
to produce peptones, Peptones are relatively more expensive, hence not widely used in industries.

2.0 MEDIA STERILIZATION

2.1 Culture Media Sterilization

The components of culture media, water, and containers all contribute to the contamination of vegetative
cells and spores. Before using in fermentation, the media must be free of contamination (Marcel et al, 2010).
Heat is the most prevalent way for sterilizing media; however other methods are used to a lesser extent
(physical methods, chemical treatment, and radiation).

1. Sterilization by heat:
Boiling >100°C for 20-50 minutes, Autoclaving; 121°C for 15-20 minutes, Dry heat; 160°C for 2 hours or 170°C
for 1 hour are all viable. Heat is the most extensively used sterilization method, particularly saturated steam
under pressure (Autoclaving) or hot air sterilization, which are both traditional and reliable. Bacterial
endospores are the most thermos resistant of all cells, so destroying them usually ensures sterility. Heat
sterilization success is influenced by the quality and quantity of contamination (i.e., the kind and load of
microorganisms), the composition of the media and its pH, and the size of the suspended particles. Vegetative
cells are generally killed in a short time at a lower temperature (about 60°C in 5-10 minutes) (Sridhar,
2021).The elimination of spores, on the other hand, necessitates a greater temperature and a longer duration
(about 80°C for 15-20 minutes). The most heat resistant spores are those of Geobacillus stearothermophilus
(Wells-Bennik, 2019). This bacterium is actually used to check the sterility of fermentation equipment. These
biological markers, which come in the form of spores in glass vials with liquid media or spores on strips of
paper within glassine envelopes, are placed in places where steam is difficult to reach to ensure that steam is
penetrating.

-Mechanism.
Thermal/heat sterilization exploits a microorganism's thermal liability to prevent it from growing. Microbes
are killed by heat because their enzymes and proteins are denaturated (Sridhar, 2021).
Microorganisms are killed by moist heat because proteins coagulate and are destroyed. Then sterilization,
followed by cooling for 1-2 hours, followed by another 20-60 minutes for the actual sterilization procedure
(William, 2008).
Industrially
In a bioreactor, the autoclave mediums are heat sterilized at 121°C in batch quantities and 140°C in
continuous sterilization. The contents of the bioreactor take a few hours (2-4 hours) to reach the required
temperature (120oC). Another 20-60 minutes for the actual sterilization process (Sridhar, 2021).

6
-Batch sterilization:
Steam can be injected into the inside coils of a batch sterilizer (indirect approach). The culture media are
sterilized in batches at 121 degrees Celsius. It's quite costly, and it wastes a lot of energy (Jha, 2021).

-Continuous sterilization:
Continuous sterilization is accomplished by infusing steam directly into the system or via a heat exchanger. In
either case, the temperature is swiftly increased to 140°C and held for 30 to 120 seconds (2minutes). Three
types of heat exchangers are employed in the continuous sterilizing process: the first heat exchanger elevates
the temperature from 90 to 120 degrees Celsius for 20 to 30 seconds.
The second heat exchanger boosts the temperature to 140 degrees Celsius and keeps it there for 30 to 120
seconds.
The third heat exchanger cools the system in the next 20–30 seconds, lowering the temperature.
Because of the several steps involved in continuous sterilization, such as exchanger, heater, heat maintenance
unit, recovery of residual heat, cooling, and fermenter, roughly 80 to 90 percent of the energy is conserved.
Although a single 100ml takes 12 minutes to reach 121 when placed in a crate with other bottles, it takes 19
minutes to reach 121 when placed in the center of stacked crates (Jha, 2021).
Nutrients are destroyed when complex media containing peptides, carbohydrates, minerals, and metals are
heated. Denaturation of heat labile chemicals, such as vitamins and antibiotics, occurs as a result of either
direct heat breakdown or a reaction between medium components.

As a result, it's critical to optimize the heating process as a whole, acknowledging that short-duration, high-
temperature procedures are more fatal to microorganisms and less chemically harmful than longer-duration,
lower-temperature processes. For example, 3 minutes at 134 degrees is superior to 20 minutes at 115
degrees and 10 minutes at 126 degrees.

Sterilizing media in glass bottles in volumes (ml) and periods (minutes), such as;
19 minutes for 100ml
18 minutes for 500ml
27 minutes at 121°C for 2 liters
and 37 minutes at 121°C for 5 litres (5000ml)

To avoid overheating big volume media units, the "heat up" and "cool down" times are usually incorporated
within the 121oc holding time. Because autoclaves vary in performance, thermocouple experiments using
different quantities of media should be performed to assess the heat up and cool down times (Sridhar, 2021).

There are four phases to the sterilizing process:

The chamber heat-up time is measured with a recording probe placed in the air-discharge value found in the
chamber's base.
Stage 1: The chamber heat-up time (20-120 degree Celsius) is measured with a recording probe positioned in
the air-discharge value located in the chamber's base.
Stage 2: The heat penetration time of the medium containers is measured with thermocouples in the center
of the inner-most container at temperatures ranging from 100 to 121 degrees Celsius.
Stage 3: The holding time at the specified temperature is 121oC- 121oC (Stanbury, 2017).

7
The following are the recommended holding times:
Temperature(oC) 121 126 134

Time (minutes) 20 10 3

Important factors that influence the success of heat sterilization include;

 The quality and quantity of contamination are two important criteria that determine the success of
heat sterilization (the extent of the load and type of any contamination)

a. The nature of the goods, i.e. the media's content of heat labile substances
b. The particle size of the suspended matter
c. The media's pH level
d. The conditions in which the final product was created.
It is necessary to follow the guidelines for excellent manufacturing practice. Whatever sterilizing method is
chosen, it must be validated for each type of product or material, both in terms of sterility assurance and in
terms of cost, to guarantee that no negative changes have occurred within the product. Failure to adhere to a
stated, proven procedure to the letter could result in a contaminated or deteriorating product (Jha, 2021).

2. Filtration;
This is the most widely and frequently used method for sterilizing animal cell cultures. Certain components of
culture media (vitamins, blood components, antibiotics) are heat labile and thus destroyed by heat
sterilization; these components of the medium are totally dissolved and then filtered sterilized. They will be
removed along with germs if they are not completely dissolved (Sridhar, 2021).
Note; that air sterilization by filtration is the most prevalent method, but air sterilization by heat is no longer
employed because to its high cost.

3. Radiation;
Sterilization is most commonly used on dry items like surgical instruments and pharmaceutical products.

There are two types of radiation:

a. Ionizing Radiation
b. Non Ionizing Radiation

Sterilization by Irradiation;
Gamma radiation is frequently used to disinfect plate media used in clean rooms (ionizing radiation). When
supplied to the sterility test area double wrapped, bottled media, such as that used for sterility testing, can be
irradiated. High-energy radiations released by certain radioactive isotopes are known as gamma radiation.
Gamma radiation has a high penetrating power and a severe microbicide impact, striking critical microbial cell
components such as DNA and producing ionization, resulting in microbial cell death. (Jha, 2021).

To destroy microorganisms, the radiation dose must be chosen to be sufficiently high. Microorganisms, unlike
more complex organisms, can regenerate from a single surviving cell; additionally, individual cells of some
microbial species are highly resistant to the lethal effects of radiation, exceeding the resistance of other
biological species by orders of magnitude (such as Deinococcus radiodura) (Formerly micrococcus
8
radiodurans) (William, 2008). As a result, radiation dosages much exceeding those deadly to animals must be
used to assure that no viable organisms remain to repopulate. The most common method of gamma
irradiation for media is to employ Cobalt-60 as the radiation source. For prepared culture medium, a dose of
15 to 25 KGy is usually sufficient, while some manufacturers have utilized doses as high as 40 KGy for fried
powders. Unlike steam sterilization, the method for calculating the dose is based on determining the media
bioburden's radiation sensitivity (here radiation resistant biological indicators are not used).After determining
sensitivity to a low radiation dose and quantifying the mean bioburden, a statistically calculated larger dose is
used to give the requisite sterility assurance safety margin. After an initial evaluation, gamma dosage is
measured in each batch using DOSIMETERS, which allow for parametric release (Jha, 2021).

Radiation dosages that are either high or too low might change the pH of certain media, resulting in a
softening of the material (the outcome of partial degradation of the polymeric structures of the afar). Other
media, such as MacConkey what and CLED (Cysteine Lactose Electrolyte Deficient) media, require additives to
make them radiation sterilizable, such as the addition of sodium thioglycollate as a radioprotectant to
MacConkey what and CLED (Cysteine Lactose Electrolyte Deficient) media (The later medium is used in clinical
Microbiology to screen for urinary tract infections).

However, care must be given while choosing the dose and time for irradiation so as not to destroy the
chemicals in the Media and render it useless. Vital to look at the medium's growth-promoting qualities both
before and after irradiation when determining the dose is required. Ultraviolet light, which is more commonly
used for cell culture media, is an alternative to gamma radiation. According to yen and colleagues, UV light's
method of action is non-thermal and non-adulterating (Marcel, 2010). Wavelengths of about 2650 nm So
have the best bactericidal efficacy where nucleic acids absorb the most ultraviolet light, resulting in the
creation of pyrimidine dimers that limit microbial DNA replication. However, because ultraviolet light has a
low penetrating power, it is not always appropriate. A second area is electron beam radiation, which is used
when the gamma sterilization process takes too long or causes some media components to degrade
(Stanbury, 2017).

4. Chemical Sterilization;
Sterilants are gases or liquids that are used to sterilize, pasteurize, or disinfect materials that are susceptible
to other methods such as radiation (gamma, electron beam, X-ray), heat (wet and dry), or other chemicals
(Sridhar, 2021). It works with a wide range of materials.
a. Nitrogen dioxide (NO2) is a sterilant gas that may be used to kill a wide variety of microorganisms, including
common bacteria, viruses, and spores.
b. Ozone – This is a disinfectant for surfaces and is used in industrial settings to sanitize water and air.
It has the advantage of being able to oxidize the vast majority of organic materials. However, because it is a
poisonous and unstable gas that must be manufactured on site, it is unsuitable for many applications.
c. Glutaraldehyde and formaldehyde- These solutions (also known as fixatives) are acceptable liquid
sterilizing agents if the immersion time is long enough.
d. Hydrogen Peroxide- Another chemical sterilizing agent is hydrogen peroxide, which comes in two forms:
liquid and vaporized hydrogen peroxide (VHP). Hydrogen peroxide is a powerful oxidant that may kill a wide
spectrum of pathogens.
e. Ethylene oxide: This is the most common chemical used to sterilize, pasteurize or disinfect items that are
sensitive to processing with other methods such as radiation (gamma, electron beam, X-ray), heat (moist and
dry), or other Chemicals. It has a wide range of material compatibility.

9
Heat and filtration are the most often utilized sterilizing technologies in industry. Filtration and heat
sterilization are sometimes used in tandem or combination. For example, the water used to prepare the
media is filtered, while the concentrated nutrient solution is heat sterilized. The water is now added to dilute
the medium properly.

Chemical methods (disinfectants) and radiation procedures (U.V. rays, y-gamma) are both used but not
commonly.

5. Physical Methods:
Filtration, centrifugation, and adsorption (to ion-exchangers or activated carbon) are some of the physical
processes used. Filtration is the most widely employed of these. Heat sterilization destroys certain
components of culture media (vitamins, blood components, antibiotics) because they are heat labile. Such
medium components are totally dissolved (absolutely necessary or else germs would be removed) and then
subjected to filter sterilization (Stanbury, 2017).
The filtration approach has a handful of drawbacks:
1. High-pressure filtering is unsuitable for use in industries.
2. Some media components may be lost in the process.

Filtration and heat sterilization are sometimes used in tandem. For example, the water used to prepare the
media is filtered, and the concentrated nutrient solution is heat sterilized. The filtered water is now added to
the media for proper dilution. For media sterilization, chemical methods (using disinfectants) and radiation
processes (using UV rays, y rays, and X-rays) are not widely utilized (Bansod, 2021).

6. Batch Sterilization;

Batch sterilization: In the bioreactor, the culture mediums are sterilized in batch volumes at 121°C. Steam can
be injected directly into the medium (direct approach) or into the inner coils (indirect method) for batch
sterilization (indirect method). Steam should be pure and free of all chemical additions for direct batch
sterilization (that usually come from steam manufacturing process).

Batch sterilization has two major drawbacks:

1. Negative effects or Damage to culture media:
Changes or Alteration in nutrients, pH, and discoloration of the culture media are all typical.
2. Excessive energy use:
The total contents of the bioreactor need a few hours (2-4 hours) to reach the required temperature (120°C).
The real sterilization procedure will take another 20-60 minutes, followed by a chilling period of 1-2 hours.
Because this entire procedure wastes energy, batch sterilization is highly expensive (Jha, 2021).

7. Continuous Sterilization:

Continuous sterilization takes place at a temperature of 140°C for 30 to 120 seconds. (This is in contrast to
batch fermentation, which takes 20-60 minutes at 121°C.) This is based on the idea that at higher
temperatures, the time necessary to destroy microbes is substantially less. Continuous sterilization is carried
out by directly injecting the steam or by means of heat exchangers.

Continuous sterilization is accomplished by infusing steam directly into the system or via heat exchangers.

10
In either case, the temperature is rapidly increased to 140°C and held for 30-120 seconds. Exchanger, heater,
heat maintenance unit, residual heat recovery, cooling, and fermenter are the various steps. Three types of
heat exchangers are employed in the continuous sterilizing process. Within 20-30 seconds, the first heat
exchanger boosts the temperature to 90-1 20°C. The second exchanger boosts the temperature to 140°C and
keeps it there for another 30-120 seconds. The third heat exchanger cools the system in the next 20-30
seconds, lowering the temperature. The amount of time it takes to sterilize is based on the size of the
particles suspended.

The larger the object, the longer it will take to complete (Stanbury, 2017). The main benefit of continuous
sterilization is that it saves roughly 80-90 percent of the energy. However, because to extremely significant
temperature variations that occur in a relatively short time between sterilizing and cooling, certain chemicals
in the medium precipitate (e.g., calcium phosphate, calcium oxalate). Continuous sterilization makes starch-
containing culture media viscous, therefore it is not employed (Jha, 2021).

8. Sterilization of Air:
Industrial fermentations are typically carried out with intense and constant aeration. The air must be fully
sterile and free of any microorganisms and suspended particles for a successful fermentation. The amount of
suspended particles and microorganisms in the ambient outside air varies greatly.
Microorganisms can be found in concentrations of 10-2,000/m3, while suspended particles can be found in
concentrations of 20-100,00/m3. Fungal spores (50 percent) and Gram-negative bacteria (40 percent) are the
most common microorganisms found in the air. Filtration, heat, UV radiation, and gas scrubbing can all be
used to sterilize air or other gases. Heat and filtration are the most often utilized methods (Jha, 2021).

(a) Heat sterilization of air: In the beginning, air was sanitized by passing it over electrically heated devices.
However, because this is fairly costly, it is no longer in use.
(b) Air sterilization by filtration

In the fermentation industry, air filtration is the most often utilized sterilization method.
9. Depth Filters:
Particles are captured and removed as air passes through glass wool containing depth filters, Physical effects
such as inertia, blockage, gravity, electrostatic attraction, and diffusion are used in this filtration process.
Filters made of glass wool can be steam sterilized and reused. However, because glass wool cannot be reused,
there is a limit to how many times they can be used Glass fiber filter cartridges (which do not have the
limitations of glass wool filters) have been popular in recent years (Stanbury, 2017).

10. Filters with Membrane Cartridges:

These are pleated membrane filters constructed of cellulose ester, nylon, or polysulfone that can be removed.
Membrane cartridge filters are smaller, easier to use, and replace than other types of filters. The most
significant drawback of air sterilization is the lack of a filter capable of removing bacteriophages.
Bacteriophages have the ability to stifle industrial fermentation. Bacteriophages, for example, prevent
Corynebacterium glutamicum from producing glutamic acid (Jha, 2021).

2.1 STERILIZATION BIOLOGICAL INDICATORS

A biological indicator is a specified preparation of a certain microorganism. Spore-forming bacteria are
commonly recognized as ideal for biological indicators because, with the exception of ionizing radiation
processes, they are substantially more robust than typical microflora. A biological indicator can be used to
11
help with sterilization equipment performance qualification as well as the creation and implementation of a
validated sterilization method for a specific article (Wells-Bennik, 2019).

Types of biological indicators

-Moist Heat; Bacillus Geothermophilus spores of suitable strains are used in the wet heat sterilizing
process.Other heat-resistant spore-forming bacteria employed in the development and validation of wet heat
sterilization methods include Clostridium sporogenes and Bacillus coagulans (Stanbury, 2017).

-Dry Heat; Bacillus subtilis sp. spores are sometimes used to confirm the procedure of dry heat sterilization
(Tiburski, 2013).

-Ionizing Radiation; Bacillus pumilus spores have been used to monitor ionizing radiation sterilization
procedures, although this is an outdated method. To establish radiation processes, approaches for setting
radiation doses that do not require biological indications have been frequently adopted. Furthermore, certain
bioburden bacteria can have a higher regenerative capacity.

-Ethylene Oxide Sterilization; Spores of a Bacillus subtilis subspecies (Bacillus subtilis var. niger) are often
employed for ethylene oxide sterilization. When 100% ethylene oxide or other ethylene oxide and carrier gas
systems are utilized as sterilants, the same biological indicator method is usually used (Sridhar, 2021).

2.2 STERILIZATION CHEMICAL INDICATORS

During a sterilization cycle, chemical indicators are used to assess crucial factors (such as time, temperature,
or steam saturation). They are attached to the outside of each instrument unit or positioned on the inside
(e.g., packs, peel pouches, containers, etc.). They don't show that sterilization is effective.(Van Doornmalen,
2015).
A steam indication strip is an example of a chemical indicator that changes physically when exposed to steam.
The chemical indicator employs a chemical pellet that transforms from a solid to a liquid phase when exposed
to steam. When the material is liquid, it wicks along a paper strip and is visible via the chemical indicator's
glass (Giulia, 2017).

A chemical change is caused by one or more chemical reactions in the second type of chemical indicator. The
chemical in the indicator ink reacts with one or more of the sterilizing process' essential parameters and
undergoes a chemical reaction, altering and changing the color of the indicator ink to its endpoint
coloration.(Steris, 2019).

2.3 IMPORTANCE OF STERILIZATION

*STERILIZATION IS ESSENTIAL
1. To limit the danger of surgical equipment becoming contaminated.
2. To reduce the amount of time organisms spend growing on culture media
3. Prevent sickness by eradicating some germs
4 -It also reduces the biological changes that occur in organisms.

12
Conclusion
The complete destruction of all living organisms of microbiological media and air needed for microbial
fermentation is to ensure the sterility of the fermentation. Different methods of ensuring the sterility are well
explained thus the most widely employed for both air and media sterilization is the filtration and heat method
respectively, this is because they are most cost effective and practicable that will be of great use in the
industries.

13
View publication stats

References
Bansod, T. A. (2021). Fermentation Technology. World Journal of Pharmaceutical Research.

Costa, E. T. (2002). The Effect of Nitrogen and Carbon Sources on Growth of the biocontrol agent Pantoea Agglomerans
Strain CPA-2. Letters in Applied Microbiology.

Eric, K. A. (2011). Characterization of Barley (Hordeum Vulgare) with altered carbohydrate composition.
Saskatchewan,Saskatoon: Research Doctorate Submitted to the Department of Plant Sciences.

Giulia, G. (2017). The Facts About Steam Chemical Indicators. Milestone, issue 3.

Jha, N. (2021). Methods for Sterilization of Media and Air.

https://www.biologydiscussion.com/biotechnology/bioprocess-technology/methods-for-sterilization-of-media-
and-air-with-diagram/10102.

Kiro, M. (2010, November). The effects of different carbon sources on biosynthesis of pectinolytic enzymes by
Aspergillus Niger. Applied Technologies and Innovation, pp. 23 -29.

Marcel, G. a. (2010). Characteristics and Techniques of Fermentation Systems. Food Fermentation Biotechnology.

Matuszewska, A. (2016). Microorganisms as Direct and Indirect Sources of Alternative Fuels. DOI: 10.5772/62397.

Obi, F. U. (2016). Agricultural waste concept,generation,utilization and management. Nigerian Journal of Technology.

Rajana, S. P. (2020). Microbial fermentation and its role in quality improvement of fermented foods. Journal
Fermentation.

Sergio, P. (2012). Raw materials for industrial fermentation bioprocesses improvement. Pisa (Italy): Research Doctorate
school in Biomaterials.

Sergio, S. a. (2008). Metabolic Regulation and Overproduction of Primary Metabolites. Microb Biotechnol, 283 - 319.

Sikander, A. S. (2018). Strategies AND kinetics of Industrial Fermentation for the mass production of various primary and
secondary metabolite from microbes. European Journal of Pharmaceutical and Medical research.

Singh, R. S. (2019). Microbial Enzymes. India: https://www.researchgate.net/publication/331137254.

Sridhar, R. P. (2021, June 11). Sterilization and Disinfection. Retrieved from microrao:
https://www.microrao.com/micronotes/sterilization.pdf

Stanbury, P. a. (2017). Principles of Fermentation Technology.

Steris, H. (2019, November 4). Chemical Indicators.

Tiburski, J. (2013). Comprehensive study of the heat resistance of dried Bacillus subtilis spores. Food and Nutrition, p.
Université de Bourgogne.

Van Doornmalen, J. R. (2015). Six commercially available Class 6 Chemical.

Wells-Bennik, M. P. (2019). Heat resistance of spores of 18 strains of Geobacillus stearothermophilus and Impact of
culturing condition. International Journal of Food Microbiology, 161 -172.

William, A. a. (2008). Guideline for disinfection and Sterilization Healthcare facilities. U.S.A: Department of Health and
Human Services.

14