Basic requirement of fermentation

Fermentation
Fermentation
organism
Fermentation
media

Fermenter
Media characteristics
● Carbon Source
● Nitrogen Source
● Minerals
● Vitamins
● Oxygen
● Water
Additional requirements
● Addition of precursors
● Free from toxic components
● Excellent buffering capacity
● Avoidance of foaming
● Contamination prevention
● Simpler downstream Processing
● Appropriate viscosity to ensure mass transfer
Characteristics: Raw materials

● Easily available
● Low cost and surplus
● Sufficient supply
● It should no contain impurities
● It should contain most of the compounds
C-Source

● Molasses
● Cheese whey
● Starchy Wastewater
● Cellulosic Wastes
● Sulphite Waste liquors
● Malt extract
N-Source
● Corn steep liquor
● Ammonium Salts
● Urea
● Peptone
● Yeast extract
● Soya meal
Sterilization of Culture Media and
Gases
If not sterilised

Economic Loss
Media can
contaminated
Lower yield of
product
Inhibit product
synthesis

Alter product
For successful fermentation, it is absolutely essential to ensure:

a. Sterility of the media containing the nutrients.

b. Sterility of incoming and outgoing air.

c. Sterility of the bioreactor.

d. Prevention of contamination during fermentation.

Sterilization of bioreactor
A bioreactor can be sterilized by destroying the organisms by
heat/chemicals/radiation or sometimes by physical procedures such as filtration.
 Sterilization of media and air
IMPORTANT POINTS :

Generally fermentation media is sterilized by autoclaving that is stream under

pressure .

Some components present in the media are heat sensitive, like Vitamins,
Enzymes, amino acids such component are sterilized by using bacteriological
filters.

If the media is in large amount then media is sterilized in batches by passing

through heat retention tubes.

Use of sterilization techniques depends upon type of fermentation media used

1. Sterilization of Culture Media:

The constituents of culture media, water and containers contribute to the

contamination by vegetative cells and spores. The media must be free from
contamination before use in fermentation. Sterilization of the media is most
commonly achieved by applying heat and to a lesser extent by other means (physical
methods, chemical treatment, and radiation).
Heat sterilization:

Heat is the most widely used sterilization technique. The quality and quantity of
contamination (i.e., the type and load of microorganisms), composition of the media
and its pH and size of the suspended particles are the important factors that
influence the success of heat sterilization.

In general, vegetative cells are destroyed at lower temperature in a short time

(around 60°C in 5-10 minutes). However, destruction of spores requires higher
temperature and relatively longer time (around 80°C for 15-20 minutes). Spores of
Bacillus stearothermophilus are the most heat resistant. In fact, this organism is
exploited for testing the sterility of fermentation equipment.
Physical methods:

The physical methods such as filtration, centrifugation, and adsorption (to ion-
exchangers or activated carbon) are in use. Among these, filtration is most widely
used. Certain constituents (vitamins, blood components, antibiotics) of culture media
are heat labile and therefore, are destroyed by heat sterilization. Such components of
the medium are completely dissolved (absolutely essential or else they will be
removed along with microorganisms) and then subjected to filter sterilization.
There are a couple of limitations of filtration technique:

1. Application of high pressure in filtration is unsuitable for industries.

2. Some of the media components may be lost from the media during filtration.

Sometimes, a combination of filtration and heat sterilization are applied. For

instance, the water used for media preparation is filtered while concentrated nutrient
solution is subjected to heat sterilization. The filtered water is now added for
appropriate dilution of the media. The chemical methods (by using disinfectants) and
radiation procedures (by using UV rays, y rays, X-rays) are not commonly used for
media sterilization.
Batch sterilization:

The culture media are subjected to sterilization at 121°C in batch volumes, in the
bioreactor. Batch sterilization can be done by injecting the steam into the medium
(direct method) or injecting the steam into interior coils (indirect method). For the
direct batch sterilization, the steam should be pure, and free from all chemical
additives (that usually come from steam manufacturing process).
There are two disadvantages of batch sterilization:

1. Damage to culture media:

Alteration in nutrients, change in pH and discolouration of the culture media are

common.

2. High energy consumption:

It takes a few hours (2-4 hrs.) for the entire contents of the bioreactor to attain the
requisite temperature (i.e. 120°C). Another 20-60 minutes for the actual process of
sterilization, followed by cooling for 1-2 hours. All this process involves wastage of
energy, and therefore batch sterilization is quite costly.
Continuous sterilization:

Continuous sterilization is carried out at 140°C for a very short period of time
ranging from 30 to 120 seconds. (This is in contrast to the batch fermentation done
at 121°C for 20-60 minutes). This is based on the principle that the time required for
killing microorganisms is much shorter at higher temperature. Continuous
sterilization is carried out by directly injecting the steam or by means of heat
exchangers.

In either case, the temperature is very quickly raised to 140°C, and maintained for
30- 120 seconds. The stages of continuous sterilization process and the
corresponding temperatures are depicted in picture. The different stages are—
In the continuous sterilization process, 3 types of heat exchangers are used. The first
heat exchanger raises temperature to 90-1 20°C within 20-30 seconds. The second
exchanger further raises temperature to 140°C and maintains for 30-120 seconds.
The third heat exchanger brings down the temperature by cooling in the next 20-30
seconds. The actual time required for sterilization depends on the size of the
suspended particles. The bigger is the size, the more is the time required.

The main advantage with continuous sterilization is that about 80-90% of the energy
is conserved. The limitation however, is that certain compounds in the medium
precipitate (e.g., calcium phosphate, calcium oxalate) due to very high temperature
differences that occur in a very short time between sterilization and cooling. The
starch-containing culture media becomes viscous in continuous sterilization and
2. Sterilization of Air:

In general, the industrial fermentations are carried out under vigorous and
continuous aeration. For an effective fermentation, the air should be completely
sterile, and free from all micro­organisms and suspended particles. There is a wide
variation in the quantity of suspended particles and microbes in the atmospheric
outdoor air.

The microorganisms may range from 10-2,000/m 3 while the suspended particles may
be 20-100,00/ m3. Among the microorganisms present in the air, the fungal spores
(50%) and Gram-negative bacteria (40%) dominate. Air or other gases can be
sterilized by filtration, heat, UV radiation and gas scrubbing. Among these, heat and
filtration are most commonly used.
(a) Air sterilization by heat:

In the early years, air was passed over electrically heated elements and sterilized.
But this is quite expense, hence not in use these days.

(b) Air sterilization by filtration:

Filtration of air is the most commonly used sterilization in fermentation industries.

Depth filters:

When the air is passed through a glass wool containing depth filters the particles are
trapped and removed (Fig). This filtration technique primarily involves physical
effects such as inertia, blocking, gravity, electrostatic attraction and diffusion. Glass
wool filters can be subjected to steam sterilization and reused. But there is a
limitation in their reuse since glass wool shrinks and solidifies on steam sterilization.
In recent years, glass fiber filter cartridges (that do not have the limitations of glass
wool filter) are being used.
Membrane cartridge filters:

These are removable pleated membrane filters made up of cellulose ester, nylon or
polysulfone. Membrane cartridge filters are smaller in size, simpler for operation and
replacement. The most important limitation of air sterilization is that there is no filter
that can remove bacteriophages. Bacteriophages are capable of crippling the
industrial fermentation. e.g., bacteriophages interfere in the production of glutamic
acid by Corynebacterium glutamicum.
N0/N=V

Is known as design criteria