Bioaerosol Sampling.

Many of the experiments performed in this project necessitated the capture and enumeration of bioaerosols. This had to be carried out in a safe and reproducible manner. The following section describes the equipment and protocols used for the capture of bioaerosols.

2.4.1 Aerojet General Cyclone Operation.

The air sampling device used within these studies was an Aerojet General Cyclone

(Soham Scientific) (Decker et al, 1969, Upton et al, 1994), a modified version of the

Errington-Powell cyclone (Errington and Powell, 1969). The Aerojet General Cyclone was set up as shown in figure 2.5. The principle o f operation is that the injection of a re­ cycled scrubbing liquid into the cyclone washes off cells that have been deposited onto the inner walls and collects them in a sample pot. Continuous scrubbing is achieved by inducing a slight suction at the underflow so that the liquid is drawn into the sample pot. This is achieved using a pressure equalisation pipe; a section of flexible tubing connecting the cyclone overflow to the sample pot. Thus the concentration of particles collected increases with time.

When sampling bioaerosol, air was drawn into the cyclone by an air pump (Air Control Installations) at 360 L min“l, and the scrubbing liquid, 80 mL TRS was recirculated at 20 mL min’ l using a peristaltic pump (Watson-Marlow). Sampling was carried out in batch mode for 15 minute periods, at the end of each run the volume of collection liquid remaining was measured by weighing. After collection the sample was analysed using QPCR and/or cells counts.

2.4.1.1 Cyclone Cleaning.

The cyclone and all associated tubing was cleaned after each sample by immersion into a 1% Tego solution (Th. Goldschmidt Ltd.) for 30 minutes. To ensure that all the surfaces were thoroughly wetted the cyclone was inverted half way through the cleaning

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period. After the cleaning stage the cyclone and all tubing was rinsed out with tap water and allowed to air dry for 5 minutes.

Air outlet air inlet Recirculating liquid Injection Pressure Equilisation Pipe 65 mm Peristaltic pump sample reservoir

Figure 2.5 Schematic representation of an Aerojet General Cyclone.

2.4.2 Sampling of Fermenter Exhaust Gases.

Release o f process cells into the exhaust gas during the course o f a fermentation was measured by removing the exhaust gas filter and connecting the cyclone to the exhaust gas stream. Sampling was carried out as described in Section 2.4.1. In addition to each exhaust gas sample an additional background sample was taken (with the air filter connected) in order to provide information about the levels o f background target micro­ organisms that might be detected in room air. For background samples the cyclone was left unconnected to the exit gas line, and a 10 minute sample of room air taken. After this time, a 1 mL sample was aseptically removed from the pot. On connection to the exhaust gas stream the cyclone was run for a further 15 minutes.

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Samples obtained from the cyclone were stored overnight at 4 °C and were assayed by QPCR within 24 hours. The number o f cells released was calculated by subtracting the number o f cells collected in the background sample from the number o f cells collected in the subsequent exhaust-gas cyclone sample. An identical sampling protocol was used to examine the level o f exhaust gas containment provided by the Turbosep. The fermenter used in these studies was a 250 litre stainless steel vessel (Sinclair Fabrications, 200 L working volume) agitated by a single 6 blade Rushton turbine impeller. The experimental set up is shown schematically in Figure 2.6. Further details o f the 250 L vessel are displayed in Figure 2.7.

Air outlet T urbosep Exhaust gas/foam R ecycled liquid

Figure 2.6 Schematic representation of a Turbosep where each number refers to a

sampling position.

Chapter 2. Materials and Methods 890 105 60 177 650

Figure 2.7 Geometry o f the 250 L reactor vessel (all dimensions in mm)

The Turbosep inlet was connected directly to the exhaust gas pipe while the recycle line was connected onto a second free port on the vessel top plate. The re-cycle line was designed to pass directly into the vessel and open at a level below the liquid surface. Thus any foam re-cyeled back into the vessel would not accumulate on the broth surface.

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The degree of exhaust gas containment provided by the Turbosep was determined by measuring cell release rates both upstream (position 1 and 2 in figure 2.5) and downstream (position 3 in figure 2.5).

2.4.3 Particle Counting Devices.

The instruments used to count and size liquid and airborne particles within this project are described below.

2.4.3.1 Malvern Instruments 3600Ec Particle Sizer.

The particle size of liquid home particles was measured using a 3600Ec particle sizer (Malvern Instruments). The instrument user laser diffraction to measure the size of particles. This is done using the principal of the Fraunhofer theory in measuring and interpreting the angular distribution of light diffracted by the particles. The range of particle sizes that can be quantified is set by the choice o f filter, laser focal and beam length. The instrument was set up with a focal length of 63 mm and a beam length of 14.3 mm, with a filter for particles over the size range o f 1.2 pm to 188 pm diameter. The instrument was blanked with RO water that had been filtered through a 0.22 pm cellulose acetate filter (Whatman). The sample was then added as fine droplets to prevent the formation of bubbles until the automatic sample indicator displayed optimum. The sample was analysed by the instrument as percentage volume in each of 32 different band sizes. The results were logged to a printer.

2.4.3.2 TSI Incorporated 7450 Laser Particle Counter (LPC).

The particle size of airborne particles was measured using a LPC 7450 (TSI Incorporated) laser particle counter. Similar to the Malvern particle sizer user laser diffraction to measure the size o f particles. The system comprises a vacuum pump, LPC and printer. Air is drawn into the LPC at 5 L min ^ where it provides a real time count of particles within two size bands, greater than 0.5 pm aerodynamic diameter and greater than 5.0 pm aerodynamic diameter. The instrument was set up to count particles per litre over 1 minute sample periods and the mean results were logged to the printer.

Chapter 2. Materials and Methods