If a bioreactor also contains organisms other than those to be cultivated in it, this can reduce the product yield of the process or even lead to the formation of undesirable substances that render the entire batch unusable.

It is therefore essential that no foreign germs can enter the bioreactor or survive there. Of course, bioreactors are designed accordingly, both in terms of construction and process control.

However, if contamination should occur, it is often not easy to find the cause, since many very different circumstances can lead to contamination.

Possible causes of contamination

• Foreign germs may already be present in the culture used to inoculate the fermenter (inoculum). During inoculation, these are inevitably also transferred to the bioreactor.
• Foreign germs can survive in the growth medium to be used for the bioprocess if the medium has not been properly and therefore incompletely sterilized.
• Powders used for media preparation may contain bacterial spores that can survive at the usual sterilization temperature and even at much higher temperatures. If this is the case, new powders from another batch should be requested from the supplier.
• A fermenter system may have design defects under certain circumstances. These are, for example, dead spaces that lead to cold spots in which the sterilization temperature is not reached, to which no steam is supplied or from which steam condensate can only be insufficiently removed. Of course, Bioengineering AG can exclude such defects due to its decades of experience and its detailed knowledge of corresponding recommendations and standards, e.g. of ASME-BPE or EHEDG, in the planning and implementation of its fermenter systems.
• If a completely closed system is not used for sampling, the medium in the fermenter may be contaminated during sampling or only the sample may be contaminated. If the sample is contaminated during sampling and then used to check the cultivation in the fermenter for freedom from contamination, it is often wrongly assumed that the foreign germs found originate from the fermenter. It is therefore always necessary to differentiate precisely whether the fermenter contents or only the sample is contaminated.
• Defective, wet or dirty filters are often the cause of contamination of the reactor vessel. The condition and integrity of the supply gas filters in particular must therefore be checked regularly. It is best to use new filters for each batch.
• If filters and other peripheral components are sterilized externally in an autoclave, care must be taken to ensure that steam can penetrate the filter and the hoses connected to it during sterilization.
• If dosing lines, filters or other components are sterilely connected to the bioreactor vessel after sterilization, it is essential to ensure fault-free handling. Instead of needles, the correct handling of which requires some practice, it is better to use Stericaps or sterile crosses. With these systems, manipulation errors can be largely excluded.
• Defective or permanently deformed O-rings, dirty O-ring grooves or defective diaphragms in valves are also sources of infection. Contamination caused by these can be avoided by regular checks and proactive maintenance. Mechanical seals should also be checked regularly for leakage. With double mechanical seals, any leakage of the primary seal is often not directly visible as a leakage outside the reactor. Regular leakage tests should therefore be carried out.
• Acid and caustic dosing lines must also be sterilized. Although these substances are inherently sterile, air in the pipes, for example, can allow contaminants to enter the reactor vessel.
• Media containing larger solid particles may have to be sterilized for much longer in order to safely kill off germs inside the particles.

If the medium is sterile-filtered, the filter may be defective or its sterilization may have been carried out incorrectly.

If the medium is thermally sterilized in an autoclave, the sterilization time is often chosen too short, since in most cases the medium is not stirred during sterilization in the autoclave. However, if the medium can be stirred in the autoclave, this will significantly reduce the necessary sterilization time. The quality of the medium benefits from this.

If the medium is sterilized in situ together with the fermenter, the correct procedure to prevent the survival of foreign germs is:

• The cooling of the reflux cooler in the exhaust air section must be interrupted
• When heating up to sterilization temperature, the fermenter system must be sufficiently degassed
• Any safety valves in the sterile area should be briefly vented at sterilization temperature
• Vacuum should be avoided during cooling after sterilization

When taking samples via immersion tubes, there is always the danger that liquid from a non-sterile area outside the fermenter can flow back into it. It is always safer to take samples from places below the liquid level in the reactor vessel.

Procedure for limiting and eliminating the source of contamination

If contamination with foreign germs is detected in the process, an attempt must first be made to find the cause so that the source of the contamination can be eliminated efficiently and permanently.

In order to reduce the effort required for this as much as possible, it is advisable to proceed according to a suitable strategy. Such a strategy could look something like this:

In a first step, the fermenter is sterilized once again, e.g. filled with a suitable test medium, usually a complex medium, which allows the rapid growth of as many types of organisms as possible. Care is taken to avoid the above-mentioned errors in the procedure. For the subsequent sterility test, the temperature and pH value are adjusted and the reactor is stirred and aerated for at least 2 days, with samples being taken regularly to test sterility. It goes without saying that the above-mentioned correct procedure must also be observed when taking samples. It may be possible to proceed in two steps, firstly by not aerating and then, if sterility without aeration has been demonstrated, by continuing the test with aeration.

If the fermenter system does not remain sterile, even if the supply air filter has been replaced if necessary, it may be advisable in a next step to replace all seals that are part of the sterile boundary and also to clean the O-ring grooves. If this measure still does not result in permanent sterility of the fermentation system after sterilization of the test medium, the source of infection could be spores that survive the usual sterilization conditions. Since the sterilization temperature can only be increased to a limited extent without endangering components of the fermenter system, especially seals, the so-called tyndallisation is recommended as a further measure. This rather complex procedure according to John Tyndall involves several consecutive cycles consisting of sterilization and subsequent operation of the fermenter with a suitable complex medium that allows spores to germinate. Once the spores have germinated, the resulting manifestations of the organisms can be killed again under normal sterilisation conditions.

If contaminations still occur in the process afterwards, it is very likely that these foreign germs have been introduced as spores via components of the growth medium.

Bioengineering is also happy to offer individual support to get to the bottom of possible sources of contamination and to eliminate them as part of our service.