Programas de Transferencia Monetaria en América Latina

Most studies have been focused on the release of micro-organisms in aerosols, since, in this state the released organisms may pose a threat to health and the environment and

can not be easily detected or contained (Hambleton et al, 1992).

Aerosols are metastable suspensions of particles in gases and generally occur in dilute

multiphase flows, with mass fractions below 1 0 ^ and particulate volumes below 1 0'^.

The generic term, aerosol was first coined near the end o f the First World War and was used to describe clouds of microscopic and sub-microscopic particles in air. Aerosols are produced when a force is exerted onto a liquid. If sufficient force is exerted small droplet aerosols are formed which can provide an effective mode o f transport for a wide range of minute particles of dust, minerals, trace elements and micro-organisms attached to the surface of or incorporated into the droplet.

The properties of aerosols that are important in terms o f biosafety are the concentration of hazardous material and the particle size distribution. In terms o f micro-organisms their concentration in the culture broth will be at their greatest towards the end of a fermentation, this will be reflected in the composition o f the aerosol produced. For the majority of bio-aerosols, aerodynamic diameters are generally greater than 2 pm (Upton

et al, 1994). However, the size distribution of the aerosol is dependent on the manner in which the aerosol was produced and the nature of the liquid from which it was derived

(Szewczyk et al, 1991). Pilancinski et al (1990) described the process o f aerosol droplet

formation. The air bubble generated in the bulk o f the liquid travels upwards to the surface where it forms a hemispherical film cap above the liquid surface. The bubble stays on the surface due to equilibrium between the buoyancy and surface tension forces. As the liquid from the bubble drains due to gravity the film becomes thinner and weaker until the bubble bursts. Fragments of the broken film are released as a large

Chapter 1. Introduction

number of film droplets. As the liquid depression is filled by the surrounding liquid, a jet is created at its centre. Disintegration of this jet releases a few large jet droplets which if large enough could potentially carry process micro-organisms. Hage and Wessels (1980) quantified the number of bacteria contained in varying sizes of aerosol droplets. The authors estimated that an aerosol containing 2 x 10^ cells mL^ would produce droplets of approximately 5 pm radius that would contain 1 bacterial cell. However larger droplets of 100 pm radius would contain approximately 8000 bacterial cells. The authors suggested that the larger particle of 100 pm would sediment about 100 times faster than the smaller 5 pm particle. However, since the terminal settling

velocity is proportional to d^ the 1 0 0 pm radius particle should theoretically sediment

400 times faster than the 5 pm radius particle.

Winkler (1987) investigated the number of contaminated particles in the exhaust gas and

reported that in the fermenter headspace there are about 1 0^ contaminated particles per

m^ o f gas. Since each contaminated particle contains at least one viable organism, then this describes only the minimum number present. Additionally it has been reported that

a large portion of viable cells are not culturable (Colwell et al, 1985) and since this

report relied on the measurement of the culturable cells, which in the aerosolised state is

likely to be very low (Neef et al, 1995), then the reported numbers are likely to be an

underestimate of the total number of cells present in the exhaust gas. Neef et al (1995)

reported that less than 1% of cells collected by filtration from an aerosol were able to

grow as mini-colonies, compared to 90% culturability o f cells filtered from the suspension used to produce the aerosol.

Ferris (1995) showed that the release o f micro-organisms into the fermenter exhaust gas could be collected by the use of a sampling cyclone. In these experiments it was shown

that E.coli cells could be detected, but the quantity o f cells collected was not known

precisely as the enumeration method used (microscope cell counting) was not sensitive

enough. Noble et al (1997) extended the work o f Ferris by monitoring the release of

cells into the fermenter exhaust gas from a 2L fermenter using a sampling cyclone - quantitative polymerase chain reaction (QPCR) methodology. It was found that over the course of a 5.5 hour period, 3 x 10^ proeess cells were released, this number corresponding to less than 2p,L equivalent of fermentation broth at harvest.

Chapter 1. Introduction

Other studies on aerosols produced by fermentation have concentrated on aerosol

particle diameter rather than cell concentration in the aerosol. Pilancinski et al (1990)

showed that the aerosol size distribution in the fermenter headspace, measured using an aerodynamic particle sizer was influenced by several factors such as the air flow rate, agitation rate and the rheological properties of the broth. The number of particles released was shown to increase with both agitation and aeration rate, with a significant fraction of the measured particles (30-40%) exceeding 2pm in diameter, large enough to

carry biological material from the broth. This work was extended by Szewczky et al

(1991) who measured the aerosol size distribution in the fermenter headspace and the effect of cell growth on the change in aerosol properties. The authors observed a decrease in particle concentration with increasing bacterial growth; this change being more pronounced in the size range above 2pm. The aerosol size range was found to be

practically independent to air flow rate and agitation rate for sizes less than 2 pm.

However, for particles larger than 2pm the concentration was found to increase with both agitation rate and air flow rate. Ferris (1995) postulated that as the cell density increases it is more likely that clumping of cells in the broth will occur. Larger clumps of cells may not be lifted into the aerosol due to their size distribution not being compatible with the particle size distribution of the aerosol formed or due to the increased settling velocity. This may account for the fall in number observed by

Szewczky et al {\99\).

Huang et al (1994) attempted to monitor aerosol generation as an on-line method for

biomass monitoring in an E.coli fermentation. Since there were very few particles of

greater than 1 pm diameter detected, the aerosol size distribution in the size range 0 .1 -

1pm was measured and compared with the bacterial growth rate and rheological

properties of the broth. It was found that the exhaust gas number concentration increased during the growth phase and subsequently decreased after bacterial cell concentration had reached a stable level. Metabolic changes in the composition of the fermentation medium subsequently caused the surface tension to decrease, leading to a greater forming tendency, which in turn caused greater aerosol release.

Due to the different methods of microbial capture and enumeration employed in each of these studies, direct comparisons between observed release rates is difficult. However,

Chapter 1. Introduction

the data presented does provide a good first approximation to quantify the hazards posed by the incidental release of bioaerosols.