Sterilization
in Fermentation
If the fermentation is invaded by a
foreign microorganism, then the following consequences may occur:
1. The
medium would have to 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
4. The
contaminant may produce compounds which make extraction of the final product
difficult.
5. The
contaminant may degrade the desired product, e.g. the degradation of Beta
lactam antibiotics by Beta lactamase-producing bacteria.
6.
Contamination of a bacterial fermentation with phage result in the lysis
of the culture.
The problem of contamination may be
prevented by:
1. Using a
pure inoculum to start the fermentation
2.
Sterilizing the medium to be employed.
3.
Sterilizing the fermenter vessel.
4.
Sterilizing all materials to be added to the fermentation during the
process.
5.
Maintaining aseptic conditions during the fermentation.
Medium Sterilization
Media may be sterilized by filtration,
radiation, ultrasonic treatment, chemical treatment or heat. Heat sterilization
is generally employed. Media may be sterilized either through the batch mode or
through the continuous mode.
Batch
Sterilization
A batch sterilization process results in
the destruction of nutrients. So it will
be designed such that the required sterility is achieved with minimum loss of
nutritive quality. The highest
temperature feasible for batch sterilization is 121°C. Exposure of medium to this temperature is
kept to a minimum required time.
The batch sterilization of the medium for
a fermentation may be achieved either in the fermentation vessel or in a
separate mash cooker.
The major advantages of a separate medium
sterilization vessel are
·
One cooker may be used to serve several
fermenters and the medium may be sterilized as the fermenters are being cleaned
and prepared for the next fermentation, thus saving time between fermentations.
·
The medium may be sterilized in a cooker
in a more concentrated form than would be used in the fermentation and then
diluted in the fermenter with sterile water prior to inoculation.
·
In some fermentations, the medium is at
its most viscous during sterilization.
So the sterilization vessel will be equipped with a powerful motor and
the fermenter could be equipped with a less powerful motor.
·
The fermenter would be spared the
corrosion which may occur with medium at high temperature.
The major disadvantages of a separate
medium sterilization vessel are
·
The cost of constructing a batch medium
sterilizer is almost the same as that for the fermenter.
·
If a cooker serves a large number of
fermenters complex pipework would be necessary to transport the sterile medium
and the risk of contamination is high.
·
Mechanical failure in a cooker supplying
medium to several fermenters will result in the whole system temporarily out of
work.
Continuous
sterilization
The continuous system includes a time
period during which the medium is heated to the sterilization temperature, a
holding time at the temperature and a cooling period to restore the medium to the
fermentation temperature. The temperature of the medium is elevated in a
continuous heat exchanger and is then maintained in an insulated serpentine
holding coil for the holding period. The length of holding period depend on the
length of the coil and the flow rate of the medium. The hot medium is cooled to
the fermentation temperature using two sequential heat exchangers - the first
utilizing the coming medium as the cooling source and then using cooling water.
There are two types of continuous sterilizer,
the indirect heat exchanger and the direct heat exchanger (steam injector).
The indirect heat exchanger: The
double-spiral type and the alternating plate type.
The Indirect double spiral heat exchanger
consists of two sheets of high-grade stainless steel which is curved around a
central axis to form a double spiral. The ends of the spiral are sealed by
covers. Steam is passed through one spiral and medium through the other in
countercurrent streams.
Advantages
of the spiral heat exchanger are:
(i)
The two streams of medium and cooling liquid, or medium and steam, are
separated by a continuous stainless steel barrier and thus cross contamination
between the two streams does not occur.
(ii)
Suitable for media with suspended solids. The exchanger is self-cleaning with
low risk of sedimentation.
Indirect
plate heat exchangers consist of alternating plates through which
countercurrent streams are circulated. The plates are separated by gaskets and
there are chances of cross-contamination between the two streams. Also
suspended solids in the medium may block the exchanger, so the system is useful
for sterilizing completely soluble media only.
The
direct heat exchanger –
(i)
Very short heating up times
(ii)
suitable for media containing suspended solids
(iii)
Low capital cost
(iv)
Easy cleaning and maintenance
(v)
High steam utilization efficiency
The
disadvantages are:
(i)
Foaming may occur during heating
(ii)
Since the medium comes into direct contact with steam, the steam should be
clean and free from any additives and there should be a condense dilution step.
Sterilization
of the fermenter
If
the medium is sterilized in a separate batch cooker, or is sterilized
continuously, then the fermenter is sterilized separately. This is done by
heating the jacket or coils of the fermenter with steam and sparging steam into
the vessel through all entries, and allowing steam to exit slowly through the
air outlet. Steam pressure is held at 15 psi for about 20 minutes.
Sterilization of the feeds
A
variety of additives which are to be added to the fermentation during the
process are sterilized either using the batch or the continuous sterilization
process.
Sterilization of wastes
Waste
biomass of microorganisms must be sterilized before disposal. Sterilization may
be achieved by either batch or continuous system. After sterilization, the
effluent must be cooled to below 60°C discharging it to waste.
Filter sterilization
Suspended
solids are separated from a fluid during filtration by the following
mechanisms,
(i) Inertial impaction - The fluid will flow through the filter through the route of least resistance while due to momentum, suspended particles tend to travel in straight lines and therefore become impacted upon the filter fibres.
(ii)
Diffusion - Extremely small particles in a fluid due to Brownian motion deviate
from the fluid flow pattern and become impacted upon the filter fibres.
(iii)
Electrostatic attraction - Charged particles may be attracted by opposite
charges on the surface of the filtration medium.
(iv)
Interception - Particles larger than the filter pores are removed by direct interception.
Filters
are of two types – those in which the pores in the filter are smaller than the
particles which are to be removed and those in which the pores are larger than
the particles which are to be removed. The first type is the absolute filter or
'fixed pore filters, which are supposed to be 100% efficient in removing
micro-organisms. Second type are depth filters or non-fixed pore filters and
are composed of felts, woven yarns, asbestos pads and loosely packed fibre
glass. In fixed pore filters, interception is the major mechanism of filtration
and non-fixed pore filters remove particles by inertial impaction, diffusion
and electrostatic attraction rather than interception.
Filters
should be steam sterilized before and after operation and thus the materials
must be stable at high temperatures and the steam used for sterilization must
be free of particulate matter. Thus the steam is prior filtered through
stainless steel mesh filters rated at 1µm.
Filter sterilization of fermentation
media
Media
for animal-cell culture cannot be sterilized by heat since it contains
heat-labile proteins. Thus, filtration is used. An ideal filtration system for
the sterilization should have the following criteria:
·
The filtered medium must be free of
fungal, bacterial and mycoplasma contamination.
·
There should be minimal adsorption of
protein to the filter surface.
·
The filtered medium should be free of
viruses and endotoxins.
Absolute
filtration systems for sterilization of animal cell culture medium are
available as membrane cartridges constructed from steam sterilizable hydrophilic
material and they are fitted into steam sterilizable stainless steel modules.
Filter sterilization of air
Aerobic
fermentations require large quantities of sterile air. Air can be sterilized by heat treatment, but
the most commonly used method is filtration.
References
- Principles of Fermentaion technology, PF Stanburry, A Whittaker, SJ Hall, 1995, Butterworth Heinemann Publications
- Industrial Microbiology, Second Edition, AH Patel, Trinity press
No comments:
Post a Comment