Handling fermenters and troubleshooting

1. Handling fermenters and
trouble shooting

3. Types of contamination
Contaminants can include species of:
• Bacteria
• Mycoplasmas
• Fungi
• Yeast
• Viruses
• Immortal cell lines invading other cell cultures e.g. HeLa cells

4. How to detect contamination
• Growth occurs earlier than expected and may differ in density, color and smell,
compared to an uncontaminated culture. If checked, the rate of substrate
consumption, acid production or product formation may help confirm the source.
• Cell culture medium including phenol red dye may change color from pink to
yellow as the earliest indicator of contamination due to acid formation.
• An increase in turbidity can also give an early warning.
• For cell cultures contaminated with mycoplasma or viruses, there may be no
visual changes to look for, even if using a light microscope. Poor cell growth and
overall performance of the culture may be the only clues that a problem exists.
In any of the above mentioned cases, direct observation of sample using staining,
microscopy and test kits can all help to confirm the presence of a contaminant.

5. Sources of contamination

6. Troubleshooting contaminations of bench-top
bioreactors
Check the inoculum
• Look backwards and check that the seed train has not been contaminated. Re-plating a small
sample of inoculum on a rich growth medium might reveal a hidden “passenger”.
• Use a secure inoculation technique. The “aseptic pour” into an open port presents multiple
chances for a contaminant to get in.
• A cleaning and disinfection procedure for laboratory equipment upstream of your bioreactor can
help keep contamination from spreading via the seed culture.

7. Troubleshooting contaminations of bench-top
bioreactors
Check the sterilization process and equipment
• Check the autoclave sterilizing temperature is correct using tape or test phials.
• If possible, use an external temperature sensor for the autoclave temperature mounted in the
temperature sensor pocket of the bioreactor vessel.
• Autoclaves which apply a vacuum prior to heating up offer much higher success rates in killing
germs. Make sure ALL lines dipping to into liquid are clamped off to prevent loss.
• Dry sterilization of any item will always take longer and/or need higher temperatures than using
wet steam.
• If media bottles are sterilized in an autoclave, there must be gaps for the steam to penetrate.
Tight packing may prevent items in the center receiving the full effect.
• If contamination due to a spore-forming organism keeps coming back, even after the vessel was
autoclaved (sometimes after several days of cultivation), it is best to completely disassemble the
vessel and tubing. Repeatedly autoclave with pauses between cycles to give spores a chance to
germinate. Reassemble and then autoclave again. This makes sure, that the steam can really get
into every nook and crevice.

8. Troubleshooting contaminations of bench-top
bioreactors
Check all bioreactor components and assembly
• Clean thoroughly and remove all traces of solid material
• Make sure the vessel and port O–rings are not flattened, torn, or feathered. Sometimes a poor fit may be
the cause even if the O–ring is intact. Do not forget the O–rings on and in sensors. Replace O-rings after 10-
20 sterilization cycles to ensure a good seal
• Check reagent bottle seals and feed lines for damage.
• Check the vessel seal. If a mechanical seal is used, check the lubricant is not leaking out constantly. A
damaged seal running dry will make a loud whistling noise which is easily identified.
• Sensors with a reservoir may have a contaminant present in the electrolyte.
• An exit gas filter which is wet may allow grow back of microbes able to pass through the filter pores. Ensure
an efficient gas cooler is used and air flow rates do not go above 1.5 Vessel Volumes per Minute (VVM). This
prevents droplet entrainment in the exit gas stream.
• If flexible tubing lines becomes contaminated, replacement is the only effective way to make sure no
contamination remains.
• Preassemble as much as possible, e.g., connect tubing and media bottles. Every connection to be made after
autoclaving poses a contamination risk.

9. Topics related to in-situ sterilizable
bioreactors
Some issues relate to the construction and use of in-situ sterilizable systems, including:
• Sterilization times/temperatures not set correctly
• A pressure leak from the vessel or pipework preventing sterilization temperature being reached
• A failed or misaligned mechanical seal allowing a small gap for ingress of contaminants.
• Leakage of lubricant
• Cooling water may harbour microbes, and this could reach the vessel through a crack or gap in a
heat exchanger or a faulty valve seat.
• Use of piercing septa needs skilled handling. Re-use of septa can leave a split in the silicone.
• Steam condensate traps must be checked for correct operation i.e. only open at the correct
temperature.

10. Testing for success of steps taken to eliminate
contamination
Checking the biological input material, services, and environment
Any suspect material can be checked by Gram staining and examination under a
microscope. A Gram-positive rod such as bacillus sp. will stand out in a sample
of yeast culture, for example. Culture samples, environmental air samples and
services water can be plated out on a general enrichment medium and any
growth isolated and identified. Different growth temperatures and times may
need to be tested to ensure nothing has been missed.

11. Testing for success of steps taken to eliminate
contamination
Checking the bioreactor hardware and automated processes before use
A stainless-steel vessel can be tested using overpressure to look for leaks. A significant drop in
pressure over time shows some part of the system is not sealed properly. Filter integrity can be
tested with devices available from the filter manufacturer. The effectiveness of steam
sterilization can be tested by leaving uninoculated medium in the vessel for several hours/days.
You can then see if growth occurs under the normal operating conditions of the bioprocess.
More sampling, chemical analysis and plating during the bioprocess may detect contamination
before it is apparent in the vessel. The aim will be for a “quick kill” if contamination is present,
to minimize lost time and resources.

12. Checking the methodologies of the bioprocess to minimize contamination risk
This can be preparation of media and reagents, choice of components, sterilization methods
and operator interactions. Good record keeping allows an audit of the different steps to
discover the point in the process when contamination first occurred. Reducing manual handling
such as sampling by adding online sensors will reduce the opportunities for a contaminant to
enter the vessel. Regular checks of the work area with swabs will give confidence that the
microbial load of the environment is under control. If not, cleaning and sterilization of the
whole work area with e.g. formaldehyde may be necessary.
House services such as air and water supplies can be separately assessed and treated to close
off this route for contamination of a bioreactor. This is normally the task of Estates
Management or your equivalent. Good record keeping of all these approaches will aid the
isolation and removal of any problems quickly.

13. The main points considered are
• Contaminants cover the whole spectrum of organisms from viruses to
mammalian cells, via bacteria yeast and fungi.
• Cell culture can be especially susceptible to “hidden” contamination which
can be hard to spot and eliminate.
• Simple checks can be used for bench-scale bioreactors, with special
attention on visual checks and confirmation of sterilization procedures.
• For in situ sterilized bioreactors simple tests can reveal potential problems
before the bioreactor has been inoculated.