Analysis of aerosolization and sampling factors affecting structural integrity and viability of bacterial aerosols

Bioaerosol research often requires stable and reliable aerosol generators that could produce high particle concentrations. However, there is still a lack of information about the effect of aerosolization on the physiological status of microorganisms. Here we tested the performance of several liquid bioaerosol generators including a Collison nebulizer, a LiquidSparging Aerosolizer (LSA), a C-flow nebulizer and a newly designed Single-Pass Aerosolizer (SPA). The SPA (CH Technologies, Westwood, NJ) is an atomizer similar to the Collison nebulizer; however unlike the Collison nebulizer it does not re-circulate the bacterial suspension, i.e., the suspension goes through the aerosolization process only once. We investigated each generator’s ability to preserve the culturability of aerosolized bacteria and retain the structural integrity of cell membrane at different aerosolization pressures. In addition, the effect of the sampling method on the structural integrity of E. coli and B. atrophaeus bacteria was examined by sampling the aerosolized bacteria using filtration, impaction, impingement, and our newly developed electrostatic precipitator with superhydrophobic surface. When testing the performance of bioaerosol generators, the bacteria aerosolized by the four generators were collected using a BioSampler. The amount of cell membrane damage was termed as the Cell Membrane Damage Index, which was expressed as the ratio of cell-released 16S rRNA gene copies in sampling liquid versus the entire amount of 16S rRNA gene copies in a collected bioaerosol sample (i.e., gene copies in cell pellet plus liquid). The Damage Indices for E. coli aerosolized with the Collison and C-flow nebulizers at 40 psi were significantly higher than the corresponding Indices at nebulization pressures of 5 and 15 psi. However, the increased aerosolization pressure for LSA and SPA did not have a substantial impact on cell membrane damage. Increased culturability loss was observed for bacteria aerosolized by Collison Nebulizer and SPA with the increased aerosolization pressure, while no significant change in culturability was found for the other two generators with increased aerosolization pressure. We found that the amount of damage to bacteria during aerosolization (value of Damage Index) was statistically significantly affected by the jar material of the Collison nebulizer (glass > polycarbonate, p<0.001) and by the bacteria species (E. coli > B. atrophaeus, p<0.001). To produce an aerosol at a constant concentration of 100 bacteria/cm^3, the SPA showed the highest bacterial culturability among the tested generators. Under our test conditions, only the Collison nebulizer and SPA achieved aerosol concentrations of 1000 bacteria/cm^3. At this concentration the SPA outperformed the Collison nebulizer by inducing less cell membrane damage. These results demonstrate that the newly developed SPA could be used to produce high bioaerosol concentrations with a relatively low amount of damage to bacterial cells. When E. coli was aerosolized by the Collison nebulizer and then collected for 5 minutes by filtration (Button sampler), impaction (Anderson impactor) and impingement (Biosampler), its range of Damage Index values were highest for the impaction (0.16–0.37) and lowest for filtration (0.051–0.085). However, even less damage was observed when the bacteria were collected using our new electrostatic collector with a superhydrophobic collection surface: 0.011–0.034. These values were significantly lower (p<0.05) compared to other three sampling methods. The data indicate that the amount of cell damage during bioaerosol sampling and the resulting release of DNA can be substantial and that this should be taken into account when analyzing bioaerosol samples. The data also demonstrate that electrostatic collection enables preservation of bacterial membrane integrity.