Ferris, Lisa Elaine; (1995) Microbial population balancing for containment specification. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The aim of this project is to quantify the numbers of microorganisms released from bioprocessing equipment during its normal operation. This release can occur either in the form of a liquid or an aerosol. Experimental work has concentrated on sampling and detecting aerosol releases of microorganisms. A population balancing system which allows the consistent quantitation of a microbial aerosol has been devised. This mass balancing system consists of the release and capture of a known aerosol in a contained environment. The aerosol was produced using an atomiser or a Collison Nebuliser. This aerosol was released into the Bassaire cabinet (volume 360L) and collected using an Aerojet-General cyclone sampler. The number of microorganisms that fell to the floor of the cabinet was also enumerated using Petri dishes containing a liquid. The total number of microorganisms collected from both the air in the cabinet and from the floor were enumerated under a microscope, using a counting chamber. This mass balance was carried out using both Saccharomyces cerevisiae and Escherichia coli. The cell recovery in the cyclone, i.e. the % of those cells released that were captured, was found to depend upon the released cell concentration and the composition of the suspending medium. It was possible to consistently recover over 50% of the cells released into the cabinet. The mass balance was then carried out exactly as before, but in a larger, soft film cabinet (volume 8.3m³). This cabinet was used because it was large enough to hold a piece of bioprocessing equipment, such as an APV 30CD high pressure homogeniser. Since the aim of this investigation was to quantify release from such a piece of equipment, the cabinet that it was located in should first be calibrated. Cell recoveries in the cyclone were found to be around 10-20%. Again, these recoveries were dependent upon the released cell concentration, but found to be consistent on repetition. The Aerojet-General cyclone was then used to sample the air in the cabinet whilst the homogeniser processed a suspension of S. cerevisiae. It was not possible to detect released S. cerevisiae cells in the cyclone using the counting chamber. However, when the homogeniser was used to process a recombinant E. coli suspension, released cells could be detected in the cyclone. This was because the Polymerase Chain Reaction (PCR) could be used to detect a DNA sequence present only in the recombinant E. coli. The microbial release was dependent upon operating time and pressure. The difficulty experienced in aerosolising microorganisms from a liquid suspension was investigated further by using the cyclone to sample the headspace air above a fermenter. Released S. cerevisiae cells could not be detected using the counting chamber. However, using PCR, it was possible to detect E. coli cells in the fermenter headspace. It is suggested that S. cerevisiae cells are less easily aerosolised from a liquid surface than E. coli cells. This study shows that during the normal operation of bioprocessing equipment such as a homogeniser and small scale fermenter, microbial release can be detected at a very low level only (approx. 10⁶ E. coli cells.h⁻¹). These results have important implications for both equipment design for contained operation and GMO legislation. Some suggestions for further work which could be carried out include: alterations to the cyclone design to improve cleanability, reduce liquid losses in the air stream and to achieve more efficient washing of collected cells from the cyclone walls; a more comprehesive analysis of the particle size distribution in the aerosols; an investigation of the off-gas from a recombinant S. cerevisiae fermentation; an investigation into the effects of operating pressure on microbial release from the 30CD homogeniser and finally the application of the microbial population balancing technique developed here together with Computational Fluid Dynamics to build up a picture describing the destination of release cells in a processing environment.

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