Bioprocess Technology : SCBT 32113

Lecture 6

STERILIZATION

25 % cost goes for sterilization & maintenance of

fermentation process

G. Prabhakaran 1
 Bioprocess Technology

Upstream ---------- Fermentation ----------- Downstream

Bioreactor
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 A schematic of a
bioreactor
Presentation Outline

1. Objectives of Sterilization

2. Methods of Sterilization

3. Batch Vs Continuous Sterilization

4. Sterilization of the feeds

5. Filter sterilization of air

6. Sterilization of fermenter exhaust

air

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1) Objectives of Sterilization

Sterilization
 Sterilization : sterile means devoid of life.

 Aseptic means uncontaminated.

 When a system has organisms but only those that are supposed to be
present, the situation is aseptic.

 Mistakes in sterilization means lost batches (very seldom reworked) that

must go to disposal.

 Contamination avoidance is major concern in fermentation process

 Sterilization : Elimination of all microbes by killing (including spore-

forming bacteria) or removal and inactivation of viruses in or on a
product.
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Problems with Contamination

If a fermentation process (bioprocess) is invaded by a foreign microorganism, then

the following consequences/ damages may occur:

1) The medium support the growth of both the production organism and the
contaminant, resulting in a loss of productivity.

2) If the fermentation is a continuous one then the contaminant may ‘outgrow’ the
production organism and displace it from the fermentation.

3) The foreign organism may contaminate the final product, e.g. single-cell
protein where the cells, separated from the broth , constitute the product

4) The contaminant may produce compounds which make subsequent extraction

of the final product difficult.

5) The contaminant may degrade the desired product.

6) Contamination of a bacterial fermentation with phage could result in the lysis

of the culture.

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 Methods to avoid contamination

i) Using a pure inoculum to start the fermentation

ii) Sterilizing the medium to be employed

iii) Sterilizing the fermenter vessel & all it accessories

iv) Sterilizing all materials to be added to the fermentation

during the process

v) Maintaining aseptic conditions (sterile operations)

during the fermentation

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2) Methods of Sterilization

Medium sterilization Methods

 Media sterilized by 1) filtration, 2) radiation, 3) ultrasonic treatment, 4)
chemical treatment and 5) heat.

 Steam (source of heat) used universally for the sterilization of

fermentation media. Except animal-cell culture media.

 A fermentation medium contains mixture of components, and deleterious

reactions may occur in the medium during the sterilization process,
resulting in the loss of nutritive quality.

 Sterilization method is dictated by

a) the requirement to achieve the desired reduction in microbial content &

b) with the least loss of nutrient quality during sterilization.

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Loss of nutrient quality during sterilization

Media Chemicals: Thermolabile vs. Thermostable

1) Interactions between nutrient components of the medium

Maillard-type browning reaction which result in medium

discoloration & loss of nutrient quality.

2) Degradation of heat labile components (e.g. amino acids, vitamins)

Strategies / solutions to overcome the loss of nutrient

quality

1. First type - sterilize sugars, and /or some salts separately

2. Second type - Suitable temperature - time profile – filter sterilize

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Constraints in large scale sterilization

 It is impossible to heat a batch of many thousands of liters of broth

in a tank to a high temperature, hold for a short period and cool.

 Heating and cooling periods contributing considerably to the total

sterilization time. Cost & Time.

 The only practical method of materializing the objective of a short-

time, high-temperature treatment is to sterilize the medium in a
continuous stream.

 Batch Vs Continuous Sterilization

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3) Batch Vs Continuous Sterilization

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Heat transfer configurations for bioreactors: jacketed vessel, external coil,
internal helical coil, internal baffle-type coil, and external heat exchanger.
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 TECHNOLOGY OF BATCH STERILIZATION

Can be carried out in:

1. Fermentation vessel (in situ medium sterilization)

2. Separate Mash Cooker for medium sterilization

Advantages of separate medium sterilization;

 medium sterilized while fermenters are cleaned - less downtime

 design conditions for sterilization more severe than fermentation

Disadvantages;

 Cost
 More transfers - more pipework, more contamination risk
 All fermenters would depend on cooker - fault render plant redundant

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 Advantages of batch sterilization over continuous
sterilization

i) Lower capital equipment costs

ii) Lower risk of contamination – continuous processes

require the aseptic transfer of the sterile broth to the
sterile vessel

iii) Easier manual control

iv) Easier to use with media containing a high proportion

of solid matter

v) Batch sterilization is still used in many fermentation

plants
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 Also called high temperature short time (HTST). Generally carried
out at 140°C, whereby sterilization times of 2-3 min are sufficient 16
Design of continuous sterilization process
 Because a continuous process involves treating small increments of
medium, the heating-up and cooling down periods are very small
compared with those in a batch system.
 There are two types of continuous sterilizer which may be used
for the treatment of fermentation media:

1. indirect heat exchanger and

2. direct heat exchanger (steam injector)

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Heat / Cooling

Overall = Heating + Holding + Cooling

Direct vs. Indirect Heat Exchangers

1) Direct heating:
- Steam injector

2) Indirect Heat Exchangers:

- Spiral type heat exchanger
- Plate heat exchanger
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TECHNOLOGY OF CONTINUOUS STERILIZATION

(a) STEAM INJECTION TYPE

ADVANTAGES

 higher steam utilization efficiency

 low capital cost
 easy cleaning and maintenance
 shorter heating and cooling cycle
 superior maintenance of medium quality
 ease of scale up
 easier automatic control

DISADVANTAGES

 foaming of media
 media and steam have direct contact - chemical contamination

(b) CONTINUOUS PLATE EXCHANGER

Has longer heating and cooling period
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 Continuous sterilization processes

STEAM INJECTION TYPE

Injection sterilizers

Steam is fed directly

into raw medium, so
that temp. rises to
desired level almost
immediately.

Shorter sterilization time me

ans less thermal degradation
of medium
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Continuous Sterilization

Spiral Heat Exchangers

 The most widely used continuous sterilization system based on the

spiral heat exchangers

 The fermenter prior to sterilization of the medium by circulating hot

water through a closed circuit

 The pipe work between the fermenter and the sterilizer steam
sterilized separately.

 Heat conservation is achieved by cooling the sterile medium against

cold, incoming unsterile medium which will then be partially heated
before it reaches the sterilizer

 When designing a continuous sterilization process based on spiral

heat exchangers, it is important to consider the effect of suspended
solids on the sterilization process.

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 Typical continuous sterilization system employing
spiral heat exchangers

1 steam
2 medium
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Advantages:

1) Uniform steam requirements throughout the duration of the sterilization

2) Simplified process control
3) Shorter sterilization time means less thermal degradation of medium

Disadvantages:

1) High demand for steam in a shorter period of time than batch

2) Concentration of media becomes dilute due to steam condensation
3) Since steam is actually dispersed in media, steam must be clean to avoid
contamination
High temperatures for short times are used in preparing nutrient media for industrial fermentations and in pasteurizing milk,
because this causes less damage to biochemicals than more prolonged times at lower temperatures. This exploits the
temperature effects on activation energies because bacterial killing is affected by a temperature change more than is heat
destruction of biochemicals.

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COMPARISON OF CONTINUOUS & BATCH

ADVANTAGES OF CONTINUOUS:
 Reduction of sterilization cycle time
 Ease of scale-up
 Superior maintenance of medium quality (less destruction)
 Reduced surge capacity for steam (more efficient plant use)

ADVANTAGES OF BATCH:
 Lower capital cost (fermenter used as autoclave)
 Lower contamination risk (less transfers of liquids)
 Presence of solids (particles) less of a problem

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4) Sterilization of the feeds

Sterilization of the feeds

 A variety of additives may be administered to a fermentation during

the process and it is essential that these materials are sterile.

 The sterilization method depends on the nature of the additive, and

the volume and feed rate at which it is administered.

 If the additive is fed in large quantities, then continuous

sterilization may be desirable

 Batch sterilization of feed liquids normally involves steam

injection into the material held in storage vessels.

 Whatever the sterilization system employed, it is essential that all

ancillary equipment and feed pipe work associated are sterilizable.

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Sterilization of liquid wastes

 Waste biomass must be sterilized before disposal.

 Sterilization by either batch or continuous and must carried out

under contained conditions.

 Batch sterilization involves the sparging of steam into holding tanks,

whereas continuous processes would employ the type of heat
exchangers which have been discussed.

 Whichever method employed, the effluent must be cooled to below

60oC before it is discharged to waste.

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Filter sterilization of fermentation media
 Media for animal-cell culture cannot be sterilized by
steam because they contain heat-labile proteins.
 An ideal filtration system for sterilization of animal cell
culture media must fulfill the following criteria:

i) The filtered medium must be free of fungal, bacterial

and mycoplasma contamination
ii) There should be minimal adsorption of protein to the
filter surface
iii) The filtered medium should be free of viruses
iv) The filtered medium should be free of endotoxins

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5) Filter sterilization of air

Filter sterilization of air

 Aerobic fermentations require the continuous addition of
considerable huge quantities of sterile air

 Although it is possible to sterilize air by heat treatment, the most

commonly used sterilization process is filtration

 Fixed pore filters are widely used in the fermentation industry

 The most common construction material used for the pleated

membranes for air sterilization is PTFE *, which is hydrophobic and
is therefore resistant to wetting

 PTFE may be steam sterilized and are resistant to ammonia which

may be injected into the air stream, prior to the filter for pH control

(a) Depth filters: (b) Membrane filters: * Poly tetra fluoro ethylene 31
 Types of Membrane modules
1. Plate and frame - flat sheets stacked into an
element
2. Tubular (tubes)
3. Spiral wound designs using flat sheets
4. Hollow fibre - down to 40 microns diam. and
possibly several metres long ; active layer on
outside and a bundle with thousands of closely
packed fibres is sealed in a cylinder

The pore size for filtering bacteria, yeasts, and fungi is in the range of 0.22-
0.45 μm (filtration membranes) are most popular for this purpose.
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Tubular (tubes) Spiral wound module

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Plate Heat Exchanger (PHE)

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6) Sterilization of fermenter exhaust air

Sterilization of fermenter exhaust air

 In many traditional fermentations the exhaust gas from
the fermenter vented through relatively inefficient depth
filters

 The use of recombinant organisms and a greater

awareness of safety and emission levels of allergic
compounds, the containment of exhaust air is more
common

 Fixed pore membrane nodules are also used for this

application

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 Sterilization conveniently categorized

I. Physical methods
① Heat :
 Dry heat
 Moist heat
② Radiations
Ultraviolet radiations
Ionizing radiations
③ Filtration
II. Chemical methods (gases and liquids)
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 Alcohols ,Chlorine, Formalin
1. Chemical agents
Suitable for skin and instruments

Ultraviolet and Ionizing Radiation

2. Radiation
Suitable for sterile room and inoculation hood

Direct flaming: e.g. inoculating loop

3. Dry Heat
Hot-air sterilization :160 ℃, 2h in hot air oven

121℃ ， 30 min in autoclave

4. Moist Heat
Suitable for medium and instruments

5. Filtration
Removal of bacteria by filter medium
Used for heat sensitive materials and filtrated air

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 Moist Heat vs Dry Heat

Moist heat Dry

heat
Penetrating potency higher lower

Temp. for protein clotting lower higher

Extra heat released yes no

from condensation

Sterilizing potency: Moist heat ﹥﹥Dry

heat 40
Why is moist heat more efficient
than dry heat ?

Conductivity. Moisture conducts

the heat better than a dry system.

 Dry heat- protein oxidation

 Moist heat- protein denaturation
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Additional studies for reading

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 Air sterilization system

• Sterilization of the inlet air is undertaken to prevent

contaminating organisms from entering the reactor.
• The exit air on the other hand is sterilized not only to keep
contaminants from entering but also to prevent organisms
in the reactor from contaminating the air.
• A common method of sterilising the inlet and exit air is
filtration. For small reactors (with volumes less than 5
litres), disk shaped hydrophobic Teflon membranes
housed in a polypropylene housing is used. are used.
Teflon is tough, reusable and does not readily block.

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 Sterilisation of the air

For larger laboratory scale fermenters (up to 1000 litres), pleated membrane
filters housed in polypropylene cartridges are used.

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 Sterilisation of the air
• By pleating the membrane, it is possible to create a compact filter with
a very large surface area for air filtration. Increasing the filtration area
decreases the pressure required to pass a given volume of air
through the filter.
• Sterilization of the inlet and exit air in large bioreactors (> 10,000 litres)
can present a major design problem. Large scale membrane filtration is
a very expensive process. The filters are expensive as they are difficult
to make and the energy required to pass air through a filter can be quite
considerable.
• Heat sterilization is alternative option. Steam can be used to sterilize
the air. With older style compressors, it was possible to use the heat
generated by the air compression process to sterilize the air. However,
compressors are now multi-stage devices which are cooled at each
stage and disinfecting temperatures are never reached. 45
 Condenser
In small reactors, the exit air system will typically include a condenser.

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 Condenser
• The condenser is a simple heat exchanger
through which cool water is passed.
• Volatile materials and water vapour condense
on the inner condenser surface.
• This minimizes water evaporation and the loss
of volatiles.
• Drying the air also prevents blocking of the exit
air filter with water
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 Air sterilisation system - Positive pressure

• During sterilisation the concept of "maintaining positive pressure" will

often be used.
• Maintaining positive pressure means that during sterilisation, cooling
and filling and if appropriate, the fermentation process, air must be
pumped into the reactor.
• In this way the reactor is always pressurised and thus aerial
contaminants will not be "sucked" into the reactor.
• It is very important that positive pressure is maintained when the
bioreactor is cooled following sterilisation. Without air being
continuously pumped into the reactor, a vacuum will form and
contaminants will tend to be drawn into the reactor.

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 Air sterilisation system -
Positive pressure
Maintaining positive pressure at all stages of the fermentation setup and
operation is an important aspect of reducing the risk of contamination

Without aeration, a vacuum With aeration, positive pressure

is always maintained and
forms as the reactor cools.
contaminants are pushed49away
from the reactor
 Toxins
Provide properties to spread and cause damage to the host.
Compare endotoxins and exotoxins

1. Endotoxins from inside the cell. Released upon cell lysis.

2. Exotoxins are secreted out of the cell during cell life.

• Toxin Substances that contribute to

pathogenicity
• Toxigenicity Ability to produce a toxin
• Toxemia Presence of toxin the host's blood
• Toxoid Inactivated toxin used in a vaccine
• Antitoxin Antibodies against a specific toxin
 EXOTOXIN ENDOTOXIN
1. Released from the cell before 1. Integral part of cell wall
or after lysis
2. Endotoxin is LPS; Lipid A is
2. Protein toxic component
3. Heat labile 3. Heat stable
4. Antigenic and immunogenic 4. Antigenic; ??immunogenicity
5. Toxoids can be produced
5. Toxoids cannot be produced
6. Specific in effect on host
6. Many effects on host
7. Produced by gram-positive and
gram-negative organisms 7. Produced by gram-negative
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organisms only
Heat transfer configurations for bioreactors: jacketed vessel

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External coil

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Internal helical coil

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Shell and Tube Exchanger

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