STERILISATION IN

BIOPROCESSES
• The operation of a monoseptic process requires the
attainment of aseptic conditions and the subsequent
addition of the micro-organisms of choice only into the
system.

• Many bioprocesses are designed to produce aseptic

product ranges for the pharmaceutical, diagnostic and
nutraceutical industries.

• Removal or limitation of microbial contamination in

food, healthcare and medical products is also of key
importance.
Sterilisation requires either the removal of or the destruction of the micro-
organisms present. This can be achieved by:

1. Thermal: preferred for economical large-scale sterilisations of

liquids and equipment

2. Chemical: preferred for heat-sensitive equipment

• ethylene oxide (gas) for equipment
• 70% ethanol-water (pH=2) for equipment/surfaces
• 3% sodium hypochlorite for equipment

3. Irradiation: through exposure to ultraviolet, gamma and X-rays

4. Filtration
• membrane filters having uniform micropores
• depth filters of glass wool
Etc……
Air sterilisation by filtration.
This represents the removal of the micro-
organisms. Aspects of this can be extrapolated
to liquid sterilisation by filtration

Media sterilisation by heat treatment.

This represents the destruction of micro-
organisms. Aspects of this can be extrapolated
to chemical destruction.
AIR STERILISATION BY
FILTRATION
 FILTER STERILISATION OF AIR
• The most frequent source of contamination is a poorly sterilised
air stream.
• Consider a 50 000 litre bioreactor to which air is supplied at 0.1
to 1.0 vvm (volume air per vol liquid per minute).
– This requires the supply of 7 x 106 to 7 x 107 litre of air per day.
Typically, the raw air may contain 103 – 104 cells per m3 or 1 to 10
microbes per litre. Hence some 7 x 106 to 7 x 108 microbes require
removing per day.
– Many batch processes have a duration of several days while fed-batch and
continuous processes run over extended periods.
 FILTER STERILISATION OF AIR

Filtration systems typically used include:

• the fibre or depth filter
• the membrane filter.

With either system, the filter itself requires

sterilisation (typically heat sterilisation) prior to
use.
 THE DEPTH FILTER
• Fibre filters consist of a porous or fibrous packing supported by a perforated
plate and maintained at the required packing density using a perforated plate.
Typical packing materials include glass wool, glass fibre and mineral slag
wool.

• The pore size of these filters exceeds the size of the micro-organisms to be
removed. However, the random nature of the packing implies that the
passage of most microorganisms through the filter material is intercepted by
impaction onto the filter material, electrostatic effects and gravitational effects
of the filter.

• The effectiveness of the filtration occurring is based on the probability of all

microorganisms being trapped within a particular level, L.
 THE DEPTH FILTER
The removal of micro-organisms can be described by:

ln (N / N0) = K L

Where:
N = number of viable microorganisms present
N0 = initial number of viable microorganisms present
K = filtration coefficient, a function of the gas
velocity, fibre size, and size of microorganisms
to be removed
L = filter thickness
 THE DEPTH FILTER

• robust and inexpensive

• low pressure drop
• cannot operate in the presence of moisture
• remove the contaminant as a probability function, not
providing absolute removal
• channelling may occur, making the filter less effective
• depth filters are useful pre-filters, used to enhance life
of the exclusion filter and prevent increased pressure
drop across the exclusion filter.
 THE ABSOLUTE FILTER or MEMBRANE
FILTER
• a defined porosity
• absolute removal of specified particles by size exclusion,
typically 0.22 or 0.45 m pore diameter
• also called surface filters, and are arranged in membrane
cartridges
• typical materials of construction include cellulose nitrate,
cellulose acetate, vinyl polymers and polyamides
• very thin membranes (~100 m) with structural strength
provided by support material
• steam sterilisable and retain their functionality in the presence of
moisture
• can be used for the sterilisation of heat-labile fluids
 THE ABSOLUTE FILTER or MEMBRANE
FILTER
• Membrane filtration units are expensive
• These can create a high pressure drop, particularly if used to
filter ‘dirty’ air.
Pressure drop is critical as the energy requirement for air
compression for a commercial scale aerobic bioprocess may
account for as much as 25% of the production costs.
• Tests exist to ascertain the integrity of the filter:
- direct particle counting,
- the monitoring of aerosol droplets resulting from corn oil
nebulised in oil
- “grow through” tests.
THE ABSOLUTE FILTER or MEMBRANE
FILTER
HEAT STERILISATION OF
THE LIQUID MEDIUM
 THE DEGREE OF STERILISATION

The degree of sterilisation required is quantified in terms of the del

factor, :

Ñ = ln (N0 / N)
where
N = the number of viable micro-organisms remaining
N0 = the initial number of viable micro-organisms

Typically, the sterilisation process is designed such that the

probability of contamination is 1 in 1000 or less
i.e. N = 10-3.
THE DEGREE OF STERILISATION

Thermal death of E.coli Thermal death of Bacillus

stearothermophilus
 THERMAL DEATH KINETICS
At a fixed temperature, the thermal death kinetics exhibits first order
kinetics:
dN/dt = -kN
Integrating
ln (N/N0) = -k t

where
N = the number of viable micro-organisms remaining
N0 = the initial number of viable micro-organisms
k = thermal death rate constant, a measure of the
resistance of the micro-organisms to heat.
 THERMAL DEATH KINETICS

The thermal death rate constant , k, is a measure of the

resistance of the micro-organisms to heat. As such, it is a
function of temperature, following Arrhenius behaviour:

k = A exp(-E/RT)
where
A = constant
E = “activation energy” of
sterilisation
T = absolute temperature
 THERMAL DEATH KINETICS

Typical values of sterilisation constants

“Activation Sterilisation
Micro-organism energy” E constant A
(kcal/mol) (min-1)
Bacillus 67.48 4.93 x 1037
stearothermophilus
B.subtilis 68.7 9.50 x 1037
Cl. sporogens 68.7 1.66 x 1038
vegetative cells < 20 1.20 x 1021
 BATCH
STERILISATION
TEMPERATURE
VARYING
STERILISATION
PROCESSES
 TEMPERATURE VARYING
STERILISATION PROCESSES
ln (N/N0)overall = ln (N/N0)heat + ln (N/N0)hold + ln (N/N0)cool

During the temperature varying heating and cooling sections:

ln (N/N0) = A exp[-E/RT(t)]dt

For example, with heating by heat exchange, it can be shown from a heat balance
that:
ln (N/N0)heat= A exp[-E/RTs{1 + (T1-Ts)/Ts exp (-UiAit/mCp)]dt
 CONTINUOUS STERILISATION
a. DIRECT
HEATING
through
continuous steam
injection with
flash cooling

b. INDIRECT
HEATING
through heat
transfer during
heat exchange
 CONTINUOUS STERILISATION
• high rate of heat exchange
• the heating and cooling cycles are minimised (~20s)
• sterilisation caused during the heating and cooling cycles is
typically less than 2% of the total sterilisation required.

a. continuous steam injection with flash cooling; b. heat transfer during heat exchange
 CONTINUOUS STERILISATION
Advantages of high temperature-short-time (HTST) sterilisation:
• improved steam economy is found (20 – 25% of batch
requirement)
• uniformity and reproducibility of the sterilisation
process is enhanced
• bioreactor design need no longer incorporate media sterilisation.
• reduction in the damage of medium composition.
• high levels of cell destruction.

Disadvantages of high temperature-short-time (HTST) sterilisation:

• dilution of media caused by direct steam injection (typically
10% by volume),
• fouling of the heat exchange surfaces by suspended solids
• foaming caused by the fermentation media.
 Additional Readings
• https://www.sciencedirect.com/topics/engineering/batch-sterilization
• https://www.rpi.edu/dept/chem-eng/Biotech-Environ/Projects00/sterilize/batch.ht
ml
• https://www.rpi.edu/dept/chem-eng/Biotech-Environ/Projects00/sterilize/continuo
us.html