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Journal of Aerosol Science
Journal of Aerosol Science 42 (2011) 517–531
0021-85
doi:10.1
n Corr
Tel.: þ3
E-m
journal homepage: www.elsevier.com/locate/jaerosci
Aerosolisation of Escherichia coli and associated endotoxinusing an improved bubbling bioaerosol generator
Xavier Simon a,b,n, Philippe Duquenne a,b, Veronique Koehler a,b, Cecile Piernot a,b,Catherine Coulais a,b, Marie Faure a,c
a INRS—Institut National de Recherche et de Securite, Rue du Morvan, CS 60027, 54519 Vandoeuvre les Nancy Cedex, Franceb Aerosols Metrology Laboratory, Pollutants Metrology Division, Francec Pollutant Capture and Air Cleaning Process Laboratory, Process Engineering Division, France
a r t i c l e i n f o
Article history:
Received 21 January 2011
Received in revised form
21 April 2011
Accepted 2 May 2011Available online 8 May 2011
Keywords:
Experimental bioaerosol
Bubbling generator
Gram-negative bacteria
Endotoxin
Controlled aerosolisation
02/$ - see front matter & 2011 Elsevier Ltd. A
016/j.jaerosci.2011.05.002
esponding author at: INRS—Institut National
3 383508530; fax: þ33 383508711.
ail address: [email protected] (X. Simon).
a b s t r a c t
Experimental bioaerosol generators are widely used in scientific studies. However, the
choice of such a generator for a given application is made difficult by the lack of
information on the performances or limits of these systems. In this article, we venture
the assumption that a bubbling liquid generator constitutes a promising choice to
produce experimental bioaerosols with known and controlled characteristics. A gen-
erator inspired by the Liquid Sparging Aerosolizer (LSA) developed by Mainelis et al.
(2005) was used to aerosolise microorganisms by bubbling compressed air through a
bacterial suspension film.
Performances of a modified LSA-type bubbling generator were evaluated. The
generated bioaerosols were characterised, in particular in terms of concentrations,
stability over time and reproducibility. The possibility to produce controlled
airborne endotoxin concentrations from gram-negative bacterial suspensions was also
investigated.
A test rig to generate and characterise experimental bioaerosols was designed. Tests
were performed on standardised Escherichia coli suspensions, stable over time, showing
cultivable bacteria concentrations between 1.5�108 and 3.0�108 CFU mL�1. In the
operating conditions evaluated, the generator provided aerosol concentrations in the
following ranges: 4.0�105–1.0�109 Cell m�3 total bacteria, 2.5�104–2.0�107 CFU m�3
cultivable bacteria and 20–15,000 EU m�3 endotoxins. Bioaerosol properties were stable
throughout generation (180 min) and were satisfactorily reproducible between tests.
Bioaerosol generation was controllable, making these experimental bioaerosols appropriate
for various laboratory assays, including work requiring the use of airborne endotoxins at
known concentrations. Data on both the physical and biological properties of the
bioaerosol, and very complete information on the generation system’s performance were
obtained. The influences of parameters such as airflow rate, height of liquid film or
concentration of the bacterial suspension were also evaluated. These parameters can be
used to adjust the bioaerosol to the needs of the experiment.
& 2011 Elsevier Ltd. All rights reserved.
ll rights reserved.
de Recherche et de Securite, Rue du Morvan, CS 60027, 54519 Vandoeuvre les Nancy Cedex, France.
X. Simon et al. / Journal of Aerosol Science 42 (2011) 517–531518
1. Introduction
1.1. Laboratory production of test bioaerosols
Most scientific articles presenting the results of laboratory-based bioaerosol assays rely on an experimental techniqueto aerosolise microorganisms. Laboratory bioaerosol generators are used to: measure the biocollector performances,develop sampling and analysis methods, test personal or collective protective equipments, manage studies on inhalationtoxicology, study behaviour of biological aerosols in ventilation systems, etc. These cover applications in a range of fields,including hygiene in the workplace, risk evaluation and occupational disease prevention, air quality in clean rooms andrelated controlled environments or indoor air quality. As a general rule, such laboratory assays require producing abioaerosol with some or all of the following characteristics:
–
TabNon
D
A
F
F
M
S
V
4
6
6
homogeneous in a given area (chamber, tunnel, reactor, test rig, etc.);
– stable over a given time period (a few minutes to a few hours); – defined, repeatable and reproducible physical and microbiological characteristics; – with concentrations similar to those found in the real environment targeted (clean rooms, industry, indoor areas, etc.); – with as wide a range of concentrations as possible (to create conditions representative of a wide range of contamination/exposure levels);
– in controlled atmosphere (relative humidity, temperature, etc.).Microorganisms can be aerosolised using dry or wet systems (Griffiths & DeCosemo, 1994; Reponen et al., 1997) in anattempt to reproduce the phenomena and suspension mechanisms leading to their natural dispersion (Table 1). Drydispersion methods are used to generate aerosols from fungi and some bacteria (spores, dehydrated or lyophilised cells).Microorganisms can be separated from their culture medium, conditioned as a powder and dispersed into the air byconventional techniques (blowing, brushing, vibration, etc.); they can also be dispersed directly from their culturemedium, i.e. by creating airflow across the surface of a colonised agar plate to detach spores. Wet techniques, based on theaerosolisation of droplets from a liquid suspension of biological particles, are better adapted to bacteria (vegetative cells orspores), yeasts and viruses.
Choosing a generator based on the information available in the literature is currently fastidious. Data relating to thephysical properties of experimental bioaerosols (e.g. particle number concentration, size distribution, electrical charge,presence of fragments or clumps) and their evaluation over time are generally best documented. In contrast, informationon bioaerosol generator performance in terms of biological parameters and how they evolve over time is either unavailableor incomplete in most studies. Consequently, it is difficult to determine a generator’s capacity to ensure stable biologicalparameters over time or its capacity to reproduce an identical experimental bioaerosol from one day to the next. Thedifferent biological concentrations (e.g. cultivable microorganisms, total microorganisms, endotoxins) produced by agenerator or limitations to its usefulness are also incompletely known.
The performances of the chosen generator and the bioaerosol characteristics must correspond to the specific needs ofthe experiment. Test design and organisation become more complicated when the characteristics of the bioaerosolgenerated are unknown, poorly documented or poorly controlled. This can also affect robustness and relevance of testresults.
1.2. Hypotheses and objectives
Faced with selecting a generator adapted to the needs of our laboratory trials investigating prevention of occupationaldiseases linked to bioaerosol exposure, we started with the hypothesis that the Liquid Sparging Aerosolizer (LSA)developed by Mainelis et al. (2005) was a promising choice. The LSA showed notable advantages for the dispersion ofgram-negative bacteria, the primary biological agent used in our experiments.
le 1-exhaustive list of bioaerosol generators used in published laboratory studies.
ry dispersion Wet dispersion
gar tube disperser (Reponen et al., 1997) Bubbling generator (Ulevicius et al., 1997)
an method (Kanaani et al., 2009) Liquid sparging aerosoliser (Mainelis et al., 2005)
ungal spore source strength tester (Sivasubramani et al., 2004) Megahertz frequency ultrasonic nebuliser (Grundy et al., 1990)
ulti-orifice air jets/rotating substrate generator (Jung et al., 2009) Microbial suspension electrospray system (Kim et al., 2008)
wirling-flow disperser (Reponen et al., 1997) Nebuliser (Terzieva et al., 1996)
enturi bioaerosol delivery system (Macher et al., 2008) Vibrating orifice aerosol generator (Jensen et al., 1994)
00 air jets/vibration-based generator (Scheermeyer & Agranovski, 2009) Wet spinning-disc generator (Harstad et al., 1970)
-nozzle air jet generator (Lee et al., 2010)
01 angle narrow jet method (Kanaani et al., 2008)
X. Simon et al. / Journal of Aerosol Science 42 (2011) 517–531 519
The LSA is a single-pass bubbling generator where the porous disc is not submerged in the suspension. A peristalticpump continuously feeds microbial suspension onto the disc, where it settles on the upper surface to form a thin liquidfilm. The airflow delivered through the porous disc induces aerosolisation by creating fine bubbles in the liquid film. Thisair also helps dry the released droplets and transport aerosolised microorganisms and residual liquid towards thegenerator outlet. This principle of gentle bubbling aerosolisation reduces stress and damage to microorganisms comparedto nebulisation (Mainelis et al., 2001; Reponen et al., 1997; Rule et al., 2009). Moreover, biological particles and dropletssettling at the bottom of the generator container, or impacting with its walls, are not recycled. The biological particlesemitted have not therefore undergone multiple aerosol generation cycles. We also believed that the flexibility of the LSAwould make it adaptable to our applications. Several operating conditions (airflow and liquid flow rates, height of liquidfilm, composition or concentration of the suspension, porous disc characteristics, etc.) can be easily modified in an attemptto generate a bioaerosol with controlled properties.
The choice of this liquid-based generation principle was also guided by the second biological agent involved in ourexperiments, i.e. endotoxins. We hypothesised that controlled endotoxin concentrations could be obtained by dispersionof a suspension of gram-negative bacteria. Endotoxins, lipopolysaccharides present in the outer membrane of thesebacteria, are likely to be liberated during dispersion of this type of bacteria. Controlled laboratory production of airbornecompounds and toxins from microorganisms remains a challenge. Little is known about how the generators manage oraffect components such as mycotoxins, glucans and endotoxins. Few data are available on the techniques or protocolsleading to aerosolisation of endotoxins or on the concentrations obtainable.
How such an LSA-type bubbling generator would perform was, however, uncertain and required evaluation. Whileof undeniable interest, data produced by Mainelis et al. in 2005 were obtained using aerosols produced from solid(PSL or NaCl) rather than biological particles. The particle number concentration and size distribution were the onlyphysical parameters measured for a bioaerosol (Pseudomonas fluorescens) generated using this system; no analysis ofbiological parameters was performed. Further study is required to see how the LSA performs with regard to dispersion ofbiological agents.
A bioaerosol generator, based on the principle of the LSA but with specific innovative modifications was designed andproduced (Simon et al., 2009). A chamber was also constructed to condition and sample the biological particles generated,completing the experimental setup and allowing characterisation of the bioaerosols emitted by the generator. Escherichia
coli was chosen as the model microorganism.The work presented in this article characterises the performance of this modified bubbling generator. In particular
through:
–
measuring the physical and microbiological characteristics of the bioaerosol generated; – quantifying the stability (over 30–180 min) and reproducibility of these characteristics; – determining the airborne concentrations obtained (in particular in terms of cultivable bacteria and endotoxins) andensuring that they are representative of those generally detected in workplace;
– evaluating the influence of parameters such as bubbling airflow rate or liquid film height; – validating the feasibility of generating airborne endotoxins from a suspension of gram-negative bacteria.2. Material and methods
2.1. Description of the setup for bioaerosol generation and characterisation
The test rig consists of a bubbling type generator and a chamber for bioaerosol sampling (Fig. 1). It is contained within aclass II Microbiological Safety Cabinet (Thermo Fischer Scientific, HERAsafes KS18), providing a sterile atmosphere andavoiding user exposure.
The generator developed and tested was based on the principle of the LSA (Mainelis et al., 2005). Microorganisms aredispersed by bubbling compressed air through a film of microbial suspension. A peristaltic pump (Thermo FischerScientific, Masterflexs C/Ls 77122-22) feeds the suspension onto the upper surface of a calibrated porous disc at a flowrate QL. The suspension settles as a liquid film of height, Hliq. A flow of dry compressed air passes through this liquid film ata flow rate QG, where it forms bubbles that burst to produce droplets and biological particles. The droplets andmicroorganisms are then carried by the ascending airflow towards the generator outlet. Transport of the generatedparticles towards the outlet and drying of the droplets is improved by injection of entraining air at a flow rate QE. Thisentraining air is composed of a mixture of dry and humid air in variable proportions to ensure a constant relative humidity(RH) at 72% throughout the assay.
Prior to passage through the peristaltic pump, the bacterial suspension was continuously stirred using a magneticstirrer (Thermo Fischer Scientific). This prevents cell sedimentation and ensures a homogeneous liquid volume. Thecompressed air was first dried and filtered in a treatment unit (TSIs, Model 3074B), airflow rates were then adjusted usingregulated thermal mass flow metres (Brookss, Model 5850S and 5851S). 2-mm thick stainless steel porous disc (Stemm,30 mm in diameter) with a pore diameter of 1 mm were used. For each experiment, temperature and relative humiditywere continuously measured by a thermo-hygrometer (Rotronics, HygroPalm 2). The probe (Rotronics, HygroClips) was
Hliq
Cover with 4sloped orifices
Porous disc
Compressed air inlet under the porous disc
Liquid film of microbial suspension
0.2 < Q < 10 L.min5 < Q < 50 L.min
0.004 < Q < 5 mL.min10 < L < 50 cm 10 < RH < 90 %
6 thin-walled probes
QG
QE
T - RH
ΔP
Magnetic stirrer
QL
L
Bacterial suspension
Peristalticpump
10 mm
Fig. 1. Schematics of the test setup and the dispersion cell allowing a constant, reproducible liquid film height throughout a test and between tests
(adapted from Simon et al., 2009). Flow directions are indicated by arrows. The drawing is not to scale.
X. Simon et al. / Journal of Aerosol Science 42 (2011) 517–531520
introduced into the airflow so as not to perturb air stream or particle aspiration upstream of the sampling probes (Fig. 1).Before being rejected into the Microbiological Safety Cabinet, the air and generated bioaerosol were filtered through anon-line autoclavable filtration device (Whatmans, HEPA-CAPTM 75).
Three main modifications were made to Mainelis et al.’s LSA, a patent was filed to cover this invention (Simon et al.,2009):
–
the larger diameter (2.5 cm rather than 1.4 cm) generator outlet was situated just above the bubbling zone; – entrainment air was injected through 6 orifices (4 mm diameter), located in the lower part of the generator tank anddirected towards the outlet;
– an innovative porous disc cover was developed, allowing a constant liquid film height to be maintained (Fig. 1).The first two modifications improved transport of suspended particles and droplets towards the generator outlet. Thethird allowed a constant liquid film height to be maintained over a test run, and ensured reproducibility in subsequenttests. Microbial suspension was fed into the system in excess. Excess liquid was evacuated from the dispersion cell by foursloped orifices (Fig. 1). The results presented in this article were obtained with a liquid film height (Hliq) of 8 mm.
Few details of how liquid film height was managed were given in the study by Mainelis et al. (2005). However, from thestart of our tests it appeared as a key parameter influencing the airborne concentrations, their stability and reproducibility.Our attempts to operate at equilibrated input and consumption of the liquid film (in a cover without a hole) wereunsuccessful. We therefore designed and implemented the technical innovation presented in Fig. 1. Covers producingdifferent film heights demonstrated that the thinner the liquid film, the greater the concentration generated (with all otheroperating conditions stable—preliminary data not shown). A millimetre difference in Hliq, undetectable by visualinspection of the bubbling nature or position of the liquid film, results in significant modifications to the airborneconcentrations. To control the film height is a real improvement for the reproducibility of the concentration values of thebioaerosol (Section 4.5).
X. Simon et al. / Journal of Aerosol Science 42 (2011) 517–531 521
Once transported to the generator’s outlet, the bioaerosol is directed towards the sampling chamber by a 2.5 cm i.d.stainless steel straight section of variable length (L). Whatever the flow rates or relative humidity of the air used duringexperiments, theoretical drying time for droplets generated in the system is significantly lower than the average droplettransit time through the experimental setup (generatorþstraight sectionþsampling chamber; Hinds, 2001).
The test rig was completed by a sampling chamber to condition the bioaerosol before sampling (Fig. 1). This chamberwas specifically designed to characterise the bioaerosols generated, it includes a conical entrance (approximately 30 cmlong) and a cylindrical sampling zone (30 cm long and 20 cm in diameter) with six sampling probes.
Air outflow profiles were modelled using the Fluents software (Fluent, 2006) to validate chamber’s geometry and dimensions.Observation of the profiles for air speed and stream lines (data not shown) indicates that the air jet expands symmetrically in theconical area, accompanied by a progressive drop in air speed. The probe orifices are located in a measuring area where streamlines are parallel and airflow is laminar. Whatever the operating conditions (10rQGþQEr50 L min�1, 2 L min�1 sampling flowrate for each probe), the geometry of the sampling chamber favours good particle sampling and homogeneous distribution of theaerosol over the six probes (experimental evidence presented in Section 3.4). Finally, stream lines shift beyond the sampling areaand excess air evacuated through the chamber outlet does not affect upstream particle aspiration by the probes.
The experimental bioaerosol was sampled through 20 cm long, 10 mm i.d. sampling probes. Given the total possibleairflow rate (10rQGþQEr50 L min�1) and the potential sampling flow rates of the probes, sampling must be super-isokinetic. The sampling speed imposed at the aspiration orifice of each probe is higher than the upstream air speed; thestream tube converges when approaching the entrance to the probe and air speeds increase. However, theoretical modelshave shown that, for the expected particle size, the aspiration efficiency (Davies, 1977; Grinshpun et al., 1993; Su &Vincent, 2004) and the transmission efficiency of each probe (Brockmann, 2001) were adequate. In the worst-casescenarios, the combined efficiencies (aspirationþtransmission) would be 99.5%, 96.5% and 91.2% for aerodynamicdiameters of 1, 5 and 10 mm, respectively.
2.2. Preparation and analysis of the Escherichia coli suspension
The model organism E. coli was used for aerosol generation tests. This coliform gram-negative bacterium is regularlydispersed and studied in laboratory-based bioaerosol assays. It has been suggested that this bacteria could be used as oneof the standards to test bioaerosol sampling devices (Macher, 1997) and it is often selected as a challenge bioaerosol to testair cleaning devices. E. coli is also widely present in occupational settings (refuse collection, hospital, wastewater/sewagetreatment, swine house, etc.). Dispersion of this gram-negative bacterium produces endotoxin-contaminated atmospheres,one of the aspects we wished to test.
2.2.1. Preparation of the microbial suspension before aerosol generation
To prepare the vegetative cell suspension of E. coli, a strain from the Institut Pasteur collection (CIP 53.126) wassubcultured on a Petri dish containing trypticase soya agar medium (AES Chemunex) for 24 h at 37 1C. Cells recovered fromthis dish were transferred into a sterile tryptone–salt solution (AES Chemunex) in order to obtain an optical density at600 nm (OD600) of about 0.5. 2 mL of this preparation were added to 25 mL of lactose broth (AES Chemunex) beforeincubation for 24 h at 37 1C with shaking at 300 rpm (Infors HT, Minitron). Cells were recovered after washing thesuspension three times in sterile ultra-pure water. Each wash consisted of centrifuging the culture at 7000 rpm (5100g) for7 min (Sigmas, 3–18 K) and eliminating the supernatant. After washing, the cell pellet was resuspended in water in orderto obtain an OD600 of 0.3070.03 (ThermoSpectronic, Spectrophotom�etre UV/visible Helios Gamma). Preparation of thisinoculum was completed on the day of bioaerosol generation.
2.2.2. Cultivable bacteria concentration in the suspension
Serial dilutions in tryptone–salt solution were used to analyse the cultivable bacteria concentration in the suspension.Two Petri dishes (trypticase soya agar) were inoculated per dilution for three successive dilutions and incubated at 37 1Cfor 24 h. After incubation, colonies were counted on each dish containing fewer than 300 colonies (description of thecalculations can be found in NF ISO 7218 (AFNOR, 1996)). The cultivable bacteria concentration of the suspension wasexpressed as Colony Forming Units per millilitre of liquid analysed (CFU mL�1).
2.3. Bioaerosol sampling and analysis methods
2.3.1. Cultivable bacteria concentration in the bioaerosol
The generated bioaerosol was sampled by connecting a cassette (Millipores, 37-mm polystyrene 3-piece closed-facecassette) to one of the probes of the sampling chamber. For analysis of cultivable bacteria, the cassette contained a supportpad (Millipores, thick cellulose absorbent pad) and a filter (Whatmans, Nuclepores polycarbonate membrane, 0.8 mmpore size). Sampling was performed at a flow rate of 2 L min�1 with a personal sampling pump (Gilians, GilAir-3).The duration of sampling varied between 30 and 60 min, depending on tests.
All samples were analysed just after the end of the sampling. To do so, 10 mL extraction solution (tween 80 0.01%,peptone 0.1%, ultra-pure water) was introduced into the cassette before shaking for 20 min at 2000 rpm (Heidolphs,Multi-Reaxs shaker). The extract was recovered using a syringe and transferred to a clean container for further analysis.
X. Simon et al. / Journal of Aerosol Science 42 (2011) 517–531522
Analysis was as for the suspension (Section 2.2.2). The airborne cultivable bacteria concentration was expressed as ColonyForming Units per cubic metre of air sampled by the cassette (CFU m�3).
2.3.2. Total bacteria concentration in the bioaerosol
The total bacteria cell count in a sample corresponds to all the cells present, including dead cells. The following analyticalsteps are based on a previously described protocol (Rinsoz et al., 2008). Cells were counted using DAPIs (40,6-diamidino-2-phenylindole), which intercalates into the DNA double helix, preferentially in adenosine/thymine rich sequences. DAPIs
fixation was revealed by UV irradiation. Sampling and cell extraction from the cassette were identical to those described for thedetermination of the cultivable bacteria concentration. The extract obtained after shaking the cassette was recovered with asyringe and mixed with DAPIs (Sigma) for 10 min in the dark. DAPIs is used at a concentration of 0.5 mg mL�1 in sterile water.The total volume, or an aliquot, was then filtered under vacuum (Millipores, 6-place stainless steel filter holder manifold) ontoa 47 mm diameter polycarbonate membrane (Millipores, IsoporeTM black membrane filter, 0.2 mm pore size). The membranewas rinsed twice with 10 mL sterile purified water, laid on a glass slide and air-dried in the dark. Membranes were mountedbetween slide and cover slip with paraffin oil. Total bacteria on the filter were counted using a fluorescence microscope (LEICADM 2500 microscope), at 1000� magnification over 20–30 evenly spaced fields. The microscope was connected to a videocamera (colour video camera qImaging IEEE 1394) and counts were performed using specific software (Histolab 7.4.0,Microvision). The total airborne bacteria concentration generated was expressed as total cells (cultivableþnon-cultivable-þdead cells) per cubic metre of air sampled by the cassette (Cell m�3).
2.3.3. Airborne endotoxin concentration
A cassette (Millipores, 37-mm polystyrene 3-piece closed-face cassette) was connected to one of the probes. Cassettecontained a first fibreglass filter as a support pad (Whatmans, GF/B glass microfiber filter) and a second, identical, particlecollection filter. Before use, filters were heated to 250 1C for 120 min to remove pyrogen components. Sampling wascarried out at a flow rate of 2 L min�1 with a personal sampling pump (Gilians, GilAir-3), over 60 min.
Samples were analysed immediately after the end of the sampling. These analytical steps follow the directions forendotoxin assay detailed in EN 14031 (CEN, 2003). The collection filter was removed from the cassette and transferred to apyrogen-free sterile tube (Greiner) containing 10 mL sterile pyrogen-free purified water. The tube containingthe contaminated filter was shaken for 1 min at 2500 rpm with a vortex, then for 60 min at 2000 rpm (Heidolphs,Multi-Reaxs shaker). Extraction was completed by centrifugation (10 min, 2000 rpm, 4 1C; Sigmas, 3–18 K). In parallel,the second filter was removed from the cassette and the latter was reassembled using a pneumatic press. 10 mL sterilepyrogen-free purified water was added into the cassette before shaking for 60 min at 2000 rpm (Heidolphs, Multi-Reaxs
shaker). After shaking, the extract was removed using a syringe fitted with a needle (Terumos) and transferred to a sterilepyrogen-free tube (Greiner). This second step was to assay endotoxins that might have been deposited on the internalwalls of the cassette rather than on the collection filter. Endotoxins in these two extracts were then assayed by the LALkinetic chromogenic detection assay, using Kinetic-QCLs kits (Lonza Group Ltd.). The airborne endotoxin concentration isrepresentative of all the endotoxins that have been aspirated into the cassette (collection on the filterþdeposits oninternal walls). Results were expressed as Endotoxin Units per cubic meter of air (EU m�3).
2.3.4. Particle number concentration and particle size distribution of the bioaerosol
An Optical Particle Counter (OPC–Grimms, G1109) was used for continuous real-time monitoring of the particlenumber concentration and the number size distribution of the generated bioaerosol. This OPC works at a sampling flowrate of 1.2 L min�1 and classes particles according to size (‘‘optical equivalent’’ diameter, dopt) between 0.25 and 32 mm,using 31 channels. During measurement, particle counting is integrated over 6 s.
Number size distribution of the particles generated was also assessed by two other techniques:
–
Aerodynamic Particle Sizers (APS–TSITM, model 3321)APS spectrometer provides real-time aerodynamic diameter (dae) measurements of particles from 0.5 to 20 mm, using52 channels and a sampling flow rate of 5.0 L min�1.–
MultisizerTM 3 Coulter Counters (Beckman Coulter)Particles to be analysed were sampled with a 37-mm polystyrene 3-piece closed-face cassette and recovered as detailedin Section 2.3.1. One millilitre of this suspension was diluted in an electrolyte solution (Beckman Coulters, Isotons IIdiluents). The particle sizing analyser uses the Coulters principle or Electrical Sensing Zone Method (Allen, 1981; Lines,1991) and provides equivalent volume diameter (dv) measurements of particles (analysis of 100 mL, 30-mm calibratedaperture), using 100 channels.2.4. Method of characterisation of the generator performances
Depending on the tests, the generator worked continuously for between 60 and 200 min (0.2oQGo5 L min�1;QE¼20 L min�1 at RH¼5072%). A constant liquid film height (8 mm) was maintained throughout generator use. All testswere repeated at least three times.
X. Simon et al. / Journal of Aerosol Science 42 (2011) 517–531 523
2.4.1. Equivalent sampling capacity of the six sampling probes during bioaerosol generation
The aim of these tests was to experimentally verify that the six probes of the sampling chamber sampled the bioaerosolidentically. The generator was used continuously for over 180 min at a bubbling rate (QG) of 3.5 L min�1. Particle numberconcentration was followed for each probe of the sampling chamber using a Grimms G1109 (optical diameterdopt40.3 mm). To do so, a 10-min measurement was carried out on each probe successively (see Fig. 1 for probe positionson the sampling chamber) and repeated in triplicate. Each probe i (i¼1 to 6) was therefore measured three times (j¼1 to 3);each of these three measurements is characterised by an average particle number concentration over the 10 minof measurement, indicated by CN,i,j . The average concentration measured on each probe, CN,i , can be calculated from thesethree repeats:
CN,i ¼1
3
X3
j ¼ 1
CN,i,j ð1Þ
Mean particle number concentrations were analysed to determine equivalence of sampling probes by the ANOVAtest, using statistical software StatGraphics Centurion XV (version 15.2.00). P values less than 0.05 were consideredsignificant.
Measurements carried out on each of the 6 probes also allow us to determine the homogeneity of size distribution inthe bioaerosol sampled. To do so, Grimms G1109 data were used to adjust a log–normal distribution (least squaresoptimisation) and to determine the optimal number median optical diameter (dopt ) and geometric standard deviation (sg).
2.4.2. Stability of properties of the generated bioaerosol
These tests were used to evaluate generator’s capacity to maintain stable bioaerosol properties (size distribution,particle number concentration and cultivable bacteria concentration) over 180 min. For these tests, the generator operatedcontinuously for over 180 min (QG¼3.5 L min�1; QE¼20 L min�1 at RH¼5072%; Hliq¼8 mm).
2.4.2.1. Particle number concentration for optical diameters dopt40.3 mm. Particle number concentration was recorded overtime using sampling probe 1. This measurement over 180 min was repeated three times on three different days. Each of thesethree tests (j¼1 to 3) allowed the particle number concentration, CN,jðtÞ, to be measured every 6 s over 180 min. CN,jðtk Þ isdefined as the average particle number concentration over time for successive 3 min intervals (average 30 points—k¼0 to 60to describe the 180-min run). This transformation somewhat reduces random fluctuations induced by instantaneous con-centration variations during assays and, thus, allows a better view of the general trend for concentration evolution. To take intoaccount the three tests of particle number concentration evolution over time, the following ratio was calculated:
C�Nðtk Þ ¼1
3
X3
j ¼ 1
CN,jðtk Þ
CN,jðt0 Þ
!ð2Þ
A 95% confidence interval associated with each C�Nðtk Þ value, indicated IC95ðC�Nðtk ÞÞ. This characterises the dispersion of
values between the triplicate assays.
2.4.2.2. Cultivable bacteria concentration. Successive 30-min samples using closed-face cassettes were also taken todetermine how the cultivable bacteria concentration shifts over time. These samples were taken randomly from probes 2to 6; the sequence was voluntarily modified between test repeats. The cultivable bacteria concentration evolving over180 min was therefore covered by 6 (k¼0 to 5) successive 30-min steps. This test was repeated three times (j¼1 to 3)on three different days. For test j, each 30-min period can therefore be characterised by an average cultivable bacteriaconcentration, CB,jðtk Þ.
Again, the following ratio was calculated:
C�Bðtk Þ ¼1
3
X3
j ¼ 1
CB,jðtk Þ
CB,jðt0 Þ
!ð3Þ
A 95% confidence interval associated with each C�Bðtk Þ value, indicated IC95ðC�Bðtk ÞÞ. This characterises the dispersion of
values between the triplicate assays.
2.4.3. Concentration ranges for the generated bioaerosols and reproducibility
The average concentrations of total bacteria (CT ), cultivable bacteria (CB ) and endotoxin (CE ) were measured on samplescollected in parallel over 60 min. Concentration levels were modified by varying the bubbling airflow (five levelscorresponding to five QG values indicated Q1 to Q5 in ascending order). For each flow rate studied, the averageconcentration was measured over 60 min in triplicate on three different days.
X. Simon et al. / Journal of Aerosol Science 42 (2011) 517–531524
3. Results
3.1. Characterisation of the bacterial suspension
Cultivable bacteria concentration, cell size distribution and conservation of the suspension were characterised. The final stepin preparing the bacterial suspension involved adjusting OD600 to 0.3070.03. Because of this, values for cultivable bacteriaconcentrations, measured on ten different suspensions, were systematically within the range 1.5�108–3.0�108 CFU mL�1.
The number size distribution as a function of equivalent volume cell diameters in the bacterial suspension (measured withMultisizerTM 3 Coulter Counters) showed a median volume diameter of 1.13 mm and a geometric standard deviation of 1.16.
Preliminary tests (data not shown) indicated that the initial cultivable bacteria concentration and size distributionparameters were not significantly modified over four hours (with use of magnetic stirrer), even after passage through aperistaltic pump.
3.2. Size distribution of the generated bioaerosol
An example of the size distribution of airborne particles as a function of their optical diameter, measured using aGrimms G1109 is shown in Fig. 2a. A first population, corresponding to optical diameters between 0.3 and 3 mm is fullyvisible. This population presents a median optical diameter of 0.63 mm with a geometric standard deviation of 1.24. Thesedescriptive parameters vary little with test day or generation flow rate used: between 0.61 and 0.63 mm for the medianoptical diameter and between 1.21 and 1.25 for the geometric standard deviation across all the tests performed.
A second population of particles with a diameter of less than 0.3 mm is also visible in Fig. 2a. This population is notfully characterised by the Grimms G1109, which is only capable of detecting a small portion of the distribution(0.25odopto0.3 mm). Better characterisation was achieved with a TSI optical counter (Laser Aerosol Spectrometer, model3340, 0.09–7.5 mm size range, 100 particle size channels). The median optical diameter for this population was close to0.1 mm, with a wide range of diameters, up to almost 0.3 mm (data not shown).
An example of the size distribution of airborne particles as a function of their aerodynamic diameter is shown in Fig. 2b.The population presents a median aerodynamic diameter of 0.92 mm with a geometric standard deviation of 1.21.
The size distribution of the generated cells, collected and extracted from a closed-face cassette, was measured using aCoulters Multisizer 3. A median volume diameter of 1.12 mm and a geometric standard deviation of 1.15 were observed.These parameters were very close to the characteristics of E. coli cells determined in the suspension (Section 3.1).
The difference observed between the optical, aerodynamic and volume diameters is due to differences between themeasurement methods (further discussed in Section 4.2).
3.3. Definition of a real-time indicator of total bacteria concentration
We assumed that particles with an optical diameter greater than 0.3 mm are E. coli cells, whether they are cultivable,non-cultivable or dead cells. The cumulated particle number concentration with an optical diameter greater than 0.3 mm
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1010.1
dN/d
log
(dop
t) x1
0-6 (#
.L-1
)
Optical diameter dopt (μm)
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
1010.1
dN/(N
.dlo
g(d a
e)) (
-)
Aerodynamic diameter dae (μm)
21.1σμm92.0d
g
ae==
0.3 3
24.1σ
μm63.0d
g
opt
=
=
Grimm® G1109 TSITM APS 3321
Fig. 2. Size distribution for airborne vegetative E. coli cells. QG¼2.5 L min�1; QE¼20 L min�1; RH¼5072%; Hliq¼8 mm.
X. Simon et al. / Journal of Aerosol Science 42 (2011) 517–531 525
(CN(dopt40.3 mm)) therefore constitutes, on principle, a good indicator of the total bacteria concentration (CT) of thebioaerosol.
For 15 independent generation assays, we investigated the correlation between average particle number concentrations (3:7�105rCN ðdopt40:3mmÞr5:7� 108#m�3) and average total bacteria concentrations (3:5� 105rCT r1:1� 109 Cellm�3).Good correlation was obtained for those 15 independent paired samples: logðCT Þ ¼ 1:03� logðCN ðdopt 40:3mmÞ; r2
¼0.98.This indicates that real-time measurement of the particle number concentration CN(dopt40.3 mm) can be considered as a goodindicator of the total bacteria concentration in the aerosol.
Based on this result, we adapted our protocol for aerosol generation. This new method was termed ‘‘with operatorregulation’’. It involved setting a target value for CN(dopt40.3 mm). The operator then attempted to maintainCN(dopt40.3 mm) at this target value to improve stability during aerosol generation. The operator may also use the sametarget value for several different assays to improve inter-assay reproducibility. In both cases the same method was used:slight adjustment of the bubbling flow rate (QG) to maintain the target value set. To do this, the real-time optical countermeasurements were used.
The ultimate aim of this adapted method was to use a physical parameter available in real-time (the indicatorCN(dopt40.3 mm)) to better control the biological parameters which are only measurable after subsequent analyses of thesamples collected. While the aim appears to be attained for total bacteria concentrations, it remains to be validated for thecultivable bacteria or endotoxin concentrations.
3.4. Aerosol comparison over the different probes in the sampling chamber
To validate the homogeneity of sampling of the six probes, the average particle number concentration (diametersdopt40.3 mm) CN,i and the associated 95% confidence interval IC95ðCN,i Þ were measured for each probe i (Section 2.4.1).
The average particle number concentrations measured for each of the 6 probes are very close, with no more than a1330#L�1 difference between two probes (minimum: CN,3 ¼ 110,185#L�1; maximum: CN,4 ¼ 112,015#L�1; mean:112,280#L�1). For the operating conditions, the ratio between two particle number concentrations (dopt40.3 mm)sampled by two different probes is no greater than 1.2%. ANOVA analysis showed that the six values are not statisticallydifferent at a 95% confidence level (Pb0.05).
Similarly, for each of the 6 probes, the median optical diameter and geometric standard deviation of the populationwere as follows: dopt ¼ 0: 63mm and sg¼1. 23. For the size range studied, the differences observed were one-thousandth ofthe values measured; this did not justify any distinction between probes.
These results confirm that the six probes behave identically in sampling the bioaerosol in terms of particle numberconcentration and size distribution. These assays also confirm that the bioaerosol could be sampled identically by any ofthe six probes in the sampling chamber. They validate that simultaneous sampling (samples collected in parallel duringthe same generation—Section 2.4.3) can be used to determine several concentrations (CT ,CB ,CE ).
3.5. Stability of the bioaerosol over 180 min continuous aerosol generation
The stability of the particle number and cultivable bacteria concentrations in the bioaerosol was evaluated over 180 mingeneration. The total airborne bacteria and endotoxin concentrations were not part of this evaluation. The stability of bioaerosolparameters over time was evaluated both for generation without operator regulation and with operator regulation (seeSections 3.3 and 4.3). To give an idea of the extent, no intervention is generally made during the first hour of generation,subsequently four or five adjustments per hour of generation are enough to ensure adequate stability.
3.5.1. Stability of the particle number concentration (dopt40.3 mm)
How the ratio C�Nðtk Þ (time dependant particle number concentration ratio) evolves over time with or without operatorregulation is illustrated in Fig. 3. C�Nðt0 Þ¼1 indicates a perfectly stable generation.
For the first 60 min, concentration without operator regulation was relatively stable; the average C�Nðtk Þ ratio wascharacterised by variations of about 1–2% around the value of 1. In contrast, from 60 min on, an almost linear increase inthe C�Nðtk Þ ratio was observed. The results of these three assays show that an increase from the initial ratio of about 8% is tobe expected (between 3% and 13%) over 120 min. This rises to about 13% (between 8% and 18%) with generation lasting180 min.
The results of how the C�Nðtk Þ ratio evolves over time with operator regulation show a perfectly stable curve for thewhole 180-min period. The C�Nðtk Þ ratio varies by less than 1% around the value of 1 and the confidence intervals remainbelow 0.03 throughout operation (180 min).
3.5.2. Stability of the cultivable bacteria concentration
Similarly, Fig. 4 presents how the ratio C�Bðtk Þ (time dependant cultivable bacteria concentration ratio) evolved overtime with or without operator regulation. If the cultivable bacteria concentration was perfectly stable over 180 min, the6 ratios (each covering a 30-min step) would all be equal to 1¼ C�Bðt0 Þ.
For 180 min operation at a generation flow rate of exactly 3.5 L min�1 (without operator regulation), the cultivable bacteriaconcentration increases significantly over time. The average concentration measured over the first 30 min operation increases
0.9
1.0
1.1
1.2
0
Tim
e de
pend
ent n
umbe
r con
cent
ratio
n ra
tio (-
)
Time (min)
Number concentration ratio
Number concentration ratio +/- 95% confidence interval
0.9
1.0
1.1
1.2
Tim
e de
pend
ent n
umbe
r con
cent
ratio
n ra
tio (-
)
Number concentration ratio
Number concentration ratio +/- 95% confidence interval
Without operator regulation noitalugerrotarepohtiW
CN
* (t k) ±
IC
95( C
N* (t k
))
CN
* (t k) ±
IC
95( C
N* (t k
))
60 120 180 0Time (min)
60 120 180
Fig. 3. C�Nðtk Þ ratio evolution over time for 180 min bioaerosol generation, without operator regulation (left) and with intermittent operator regulation
(right). QG¼3.5 L min�1; QE¼20 L min�1; RH¼5072%; Hliq¼8 mm.
0.5
1.0
1.5
2.0
2.5
0-30
Tim
e de
pend
ent c
ultiv
able
bac
teria
co
ncen
tratio
n ra
tio (-
)
Time (min) Time (min)
0.5
1.0
1.5
2.0
2.5
Tim
e de
pend
ent c
ultiv
able
bac
teria
co
ncen
tratio
n ra
tio (-
)Without operator regulation With operator regulation
CB
* (t k)±
IC
( CB
* (t k))
CB
* (t k)±
IC
( CB
* (t k))
30-60 60-90 90-120 120-150 150-180 0-30 30-60 60-90 90-120 120-150 150-180
Fig. 4. Evolution of the C�Bðtk Þ ratio over time for 180 min bioaerosol generation, without operator regulation (left) and with operator regulation (right).
Error bars represent the 95% confidence interval of three repetitions. QG¼3.5 L min�1; QE¼20 L min�1; RH¼5072%; Hliq¼8 mm.
X. Simon et al. / Journal of Aerosol Science 42 (2011) 517–531526
almost 1.25-fold over the following hour (from 30 to 90 min operation). If generation is maintained for more than 90 min, theconcentration can increase by between 1.5- and 2-fold.
Intermittent operator regulation of CN(dopt40.3 mm) also stabilised the other concentration values. The average values forthe C�Bðtk Þ ratio were between 0.98 and 1.09; none of the values measured over the three assays was more than 20% greater orless than 1. For data acquired with operator regulation, ANOVA analysis showed that the time dependant cultivable bacteriaconcentration ratios were not statistically different from 1 (Pb0.05). We can thus conclude that the cultivable bacteriaconcentrations measured between 30 and 180 min remain stable and close to those measured during the first 30 min.
3.6. Concentration ranges for the generated bioaerosols and reproducibility
Total bacteria (CT ), cultivable bacteria (CB ) and airborne endotoxin (CE ) concentrations were measured as a function ofthe bubbling airflow (QG). Results are given in Fig. 5. The higher the bubbling airflow, the greater the generated bioaerosolconcentrations.
The results show that the generator produced satisfactory reproducible aerosol concentrations in the following ranges:
–
total bacteria: between 4.0�105 and 1.0�109 Cell m�3; – cultivable bacteria: between 2.5�104 and 2.0�107 CFU m�3; – endotoxins: between 20 and 15,000 EU m�3.
Bubbling airflow
Total bacteriaCultivable bacteria
1×105
1×104
1×106
1×107
1×108
1×109
Q1
1×1010
1
10
100
1000
10000
100000
End
otox
in c
once
ntra
tions
(EU
.m)
CE
± IC
(CE)
Tota
l or c
ultiv
able
bac
teria
l con
cent
ratio
ns
(C
ell.m
or
CFU
.m)
CT
± IC
(CT)
and
CB
± IC
(CB)
Q2 Q3 Q4 Q5
Bubbling airflowQ1 Q2 Q3 Q4 Q5
Fig. 5. Evolution of the concentration of total bacteria, cultivable bacteria (left) and endotoxins (right) in the bioaerosol as a function of the bubbling flow
rate QG. Assays carried out with operator regulation. Error bars represent the 95% confidence interval of three repetitions. QE¼20 L min�1; RH¼5072%;
Hliq¼8 mm.
X. Simon et al. / Journal of Aerosol Science 42 (2011) 517–531 527
The precise Q1–Q5 values are not specified in Fig. 5 as these values can shift between assays and, to a lesser extent,within an assay (see Section 3.3). The range of rates used to obtain the results presented in Fig. 5 were as follows (valueswere adjusted by 0.01 L min�1 increments): Q1 between 0.38 and 0.50 L min�1; Q2 between 0.59 and 0.72 L min�1;Q3 between 2.41 and 2.55 L min�1; Q4 between 3.41 and 3.51 L min�1; Q5 between 4.70 and 5.00 L min�1. Thus, the flowrates Q1–Q5 constitute ranges of variable values rather than fixed values.
4. Discussion
4.1. Characterisation of the bacterial suspension
When generating bioaerosols using wet dispersion methods, the initial microbial suspension must be carefullyprepared. Generator performance is necessarily dependant on the parameters of the suspension from which aerosols are tobe generated.
The preparation protocol used for suspension of E. coli cells was quite similar to that used in other studies (Juozaitiset al., 1994; Mainelis et al., 2005; Qian et al., 1995; Terzieva et al., 1996). Repeated washing and resuspension ofthe microorganisms in ultra-pure sterile water, rather than a saline solution, is an important step in purification of thesuspension. It also avoids the generation of residual or undesirable particles (nutritive elements, cell debris and saltaerosols). The suspension was standardised by diluting it to an OD600 of 0.3070.03. This improved reproducibility of thecultivable bacteria concentration in both the suspension and the generated bioaerosol.
The stability of the suspension over at least four hours is also important. It allows generation to be carried out over longperiods, or successive generations to be performed without concerns about how the concentration and cells sizedistribution will evolve, affecting bioaerosol stability over time, or assay repeatability throughout the day.
E. coli is a rod-shaped nonsporulating gram-negative bacteria. The length and diameter of the rods depend on thegrowth conditions of each microorganism. A suspension of E. coli can thus contain cells of differing diameters (E0.5–1 mm) andlengths (E1–5 mm); corresponding to a mean volume close to 0.7 mm3 (Brenner, 1984; Kubitschek, 1990). Our results complywith these bibliographic data as the mean E. coli cell volume in our suspensions was 0.75 mm3 (median volume diametermeasured 1.13 mm).
4.2. Size distribution of the generated bioaerosol
The size distribution of the cells generated presented a median volume diameter of 1.12 mm (Coulters Multisizer 3),a median aerodynamic diameter of 0.92 mm (Fig. 2b—TSITM APS) and a median optical diameter close to 0.63 mm(Fig. 2a—Grimms G1109). The digression observed between volume, aerodynamic and optical diameters was due todifferences between the measurement methods. The optical diameter is an estimation of the diameter based on measuringthe light intensity diffused by the particle at a given diffusion angle. Depending on the particle properties (shape factor,refraction index, state of the surface, absorbent particle, etc.), the geometric diameter can therefore (significantly) divergefrom the measured optical diameter. This observation was previously reported for another rod-shaped gram-negativebacterium, P. fluorescens. The volume diameter of these cells is expected to range between 1.1 and 1.3 mm (Palleroni, 1984)
X. Simon et al. / Journal of Aerosol Science 42 (2011) 517–531528
and the aerodynamic diameter is expected to be close to 0.8 mm. However, the median optical diameter of aerosolisedP. fluorescens was found to be �0.55–0.60 mm (LAS-X spectrometer for Juozaitis et al., 1994; Qian et al., 1995; Terzievaet al., 1996; Grimms OPC for Mainelis et al., 2005).
These values of diameters were obtained at RH 5072% but particle diameter may increase with increasing relativehumidity. For example, the diameter of an E. coli cell can increase by a factor of almost 30% when RH rises from 20% to 90%(Lee et al., 2002).
The size distribution obtained with a Grimms G1109 (Fig. 2a) indicated the presence of residual undesired particles onchannels less than 0.3 mm. These particles are, probably, residues of the lactose broth or cellular debris still present in thesuspension despite the washing steps included in the preparation protocol. Part of this could also be due to impuritiesor dissolved particles present in the water, resulting in the formation of residual particles after droplet drying (Ho et al., 1988;Krames et al., 1991).
Whatever their origins or nature, the presence of these residual particles is not a problem given that the sizedistribution of the E. coli cell population is quite discernible and identifiable.
4.3. Choice of CNðdopt40:6mmÞ as a real-time indicator of total bacteria concentration
The first population shown in Fig. 2a (0.3odopto3 mm) corresponds to aerosolised E. coli cells, while the second(dopto0.3 mm) consists of undesired particles. Defining the CN(dopt40.3 mm) indicator and use of the method described inSection 3.3 were only possible because of the non-overlapping optical diameters of these two populations. Insufficient orineffective washing of the suspension, use of a saline solution to prepare the suspension, using generators affectingmicroorganism integrity (Mainelis et al., 2005, 2001; Qian et al., 1995; Reponen et al., 1997; Terzieva et al., 1996) orstudying small-sized agents (virus, actinomycetes spores) could pose problems when applying this method. These remarksunderline how important preparation of the microbial suspension is for subsequent aerosol generation.
The size distribution of the E. coli cells aerosolised by bubbling remained unchanged for all generation assays with ourexperimental setup, regardless of duration. The use of CN(dopt40.3 mm) as an indicator of total bacteria concentration wastherefore justified.
4.4. Stability of the bioaerosol over 180 min aerosol generation
Aerosol generation without operator regulation leads to an increase in particle number and cultivable bacteriaconcentrations (see Section 3.5; Figs. 3 and 4). The following hypothesis may explain this increase: accumulation mayoccur in the volume of microbial suspension, which is the liquid film above the porous disc. Indeed, the bacteriaconcentration in this volume of liquid (see Fig. 1) could increase slowly over time through the combined effect of thepresence of dead zones and recycling of previously aerosolised microorganisms. This recycling, which was excluded in theinitial presentation of the principle of the generator, could occur because of random bursting of multiple bubbles that leadsto formation of jet droplets and film droplets (Blanchard & Syzdek, 1988; Georgescu et al., 2002; Gunther et al., 2003).A fraction of the droplets produced may not be drawn into the ascending airflow but might fall rapidly back onto theupper surface of the 4.9 cm2 liquid film (e.g. through the effect of initial speeds and directions of projection incompatiblewith aerosolisation). This hypothesis of slow accumulation in the suspension has not yet been validated, this will requirefurther assays.
Operator regulation allows the number concentration (dopt40.3 mm) to be maintained at a target value by adjustingthe bubbling flow rate QG when necessary (Section 3.3). This makes generation assays more complex, requiring regularmonitoring of the OPC’s data. It must be noted that the system dynamics are quite slow: often, no intervention is requiredover the first hour of operation and then just four or five adjustments per hour are enough to ensure satisfactory stability.Depending on the generation time necessary or the level of stability required for the experiment, the operator can choosewhether to intervene on the bubbling flow rate or to let the system run without regulation.
We hypothesised that regulation of CN(dopt40.3 mm) during generation could improve stability for cultivable bacteriaand endotoxin concentrations (Section 3.3). The results confirmed that stability of the cultivable bacteria concentrationwas greatly improved by this method (see Section 3.5.2). Stability of the endotoxin concentration has not yet been tested.
4.5. Concentration ranges for the generated bioaerosols and reproducibility
By acting on operating parameters, such as bubbling flow rate QG, the operator can choose the concentrations generateddepending on the tests to be performed. The values obtained for each concentration type range over four orders ofmagnitude. Tight control of the liquid film height and regulation of the particle number concentration (dopt40.3 mm) arecrucial to aerosol reproducibility, as shown in Fig. 5. The concentration ranges that can be generated with the system(between 2.5�104 and 2.0�107 CFU m�3 for cultivable bacteria and between 20 and 15,000 EU m�3 for endotoxins)satisfactorily cover the ranges generally measured in the workplace (Goyer et al., 2001; Haas et al., 2010; Laitinenet al., 2001; Marchand et al., 2007; Spaan et al., 2008), making these experimental bioaerosols suitable for a variety ofexperiments.
X. Simon et al. / Journal of Aerosol Science 42 (2011) 517–531 529
E. coli is a sensitive bacterium (in particular to desiccation); sampling by air filtration on a membrane may lead tounder-estimations of cultivable bacteria compared to sampling by collection in liquid (Li et al., 1999; Wang et al., 2001).Use of a cassette (practical, cost-effective) was favoured in our assays. The general conclusions from these would beunchanged using a different biocollector: the cultivable bacteria concentrations provided by the generator can reach highlevels with satisfactory reproducibility. However, using a different biocollector might lead to different values for thecultivable bacteria concentrations because of changes to biological efficiency and stress endured by the aerosolised cells.Thus, the cultivable bacteria concentrations given should be considered minimum concentrations produced by thegenerator.
Similarly, the concentrations presented in Fig. 5 can be further extended. The lower limits indicated (CT ¼ 4:0�105 Cellm�3; CB ¼ 2:5� 104 CFUm�3; CE ¼ 20EUm�3) can be further lowered by one or a combination of: reducing thegenerating airflow rate QG, increasing the ascending airflow rate QE, diluting the bacterial suspension, increasing the liquidfilm height above the porous disc Hliq. Adjustment in the opposite direction would increase the upper limits for aerosolconcentrations (CT ¼ 1:0� 109 Cellm�3; CB ¼ 2:0� 107 CFUm�3; CE ¼ 15000EUm�3).
For tests requiring a higher total airflow (e.g. 150 L min�1) the generation conditions can be modified to return to theconcentrations indicated in this article. In fact, dilution of the bioaerosol (decreased concentrations) induced by theincrease in ascending airflow QE can be compensated by increasing the bubbling airflow QG and/or reducing the liquid filmheight Hliq and/or using a more concentrated suspension.
In the tests carried out for this study, operator regulation of the CN(dopt40.3 mm) indicator was achieved byintermittent modification of the bubbling flow rate, QG, alone. The ascending airflow rate, QE, was never modified(20 L min�1). This procedure could induce slight modifications of the total rate QGþQE (�1%). But, for experimentsrequiring a perfectly stable total QGþQE airflow rate, a compromise (simultaneous modification of QG and QE) can meetboth this requirement and the need to regulate the CN(dopt40.3 mm) indicator.
The flexibility of this generator is a definite advantage: the operator can intervene on several parameters to adjust theexperimental bioaerosol to the needs of the experiment.
The results for the endotoxin concentrations generated (Fig. 5) are particularly novel. To our knowledge, no other studydescribes the capacity of a generator to produce and control airborne endotoxin concentrations in such detail. For a given QG
flow rate, the ratio between the maximal and minimal endotoxin concentrations is between 1.3 and 2.0. Reproducibilitytherefore appears adequate to allow a variety of experiments. It is important to remember that the results described here takeall endotoxins present in the cassette into account, i.e. those collected on the filter and those deposited on the inner surfaces ofthe cassette (Section 2.3.3). Significantly lower reproducibility for endotoxin concentrations was found when only the resultsfor endotoxins collected on filter were considered. This can be explained by the variability and random nature of the quantity ofdeposits on the inner walls of the cassette during sampling (Demange et al., 1990; Hendricks et al., 2009).
How relative humidity affects concentrations was not covered in this study (all tests were carried out at RH 5072%).Survival of aerosolised vegetative bacteria has been reported to decrease with decreasing RH (Rule et al., 2009; Stewartet al., 1997; Stone & Johnson, 2002; Theunissen et al., 1993; Walter et al., 1990; Wang et al., 2001). This effect on viabilitymight lead to increased aerosolised bacteria concentrations when RH is greater than 50%.
5. Conclusion
The work of Mainelis et al. (2005) describing a single-pass bubbling generator led us suppose that it was possible to usethis wet dispersion technique to produce a well-characterised and -controlled experimental bioaerosol containing E. coli
bacteria and their associated endotoxins. Tests were carried out using a novel experimental device including a modifiedbubbling generator and a sampling chamber to characterise the bioaerosols generated.
This study evaluated the performance of an improved bubbling bioaerosol generator in terms of physical and biologicalparameters of airborne particles. The results demonstrated that this generator can yield stable (180 min), reproducible total andcultivable bacteria concentrations when aerosolising a standardised suspension of E. coli cells. We also demonstratedreproducible airborne endotoxin concentrations obtained through wet bubbling dispersion of gram-negative bacteria. Betterresults were obtained when real-time data on size distribution from the OPC were used to regulate the cumulated particlenumber concentration (dopt40.3 mm). This physical parameter can be used as a real-time indicator of the total bacteriaconcentration, thus improving the stability and reproducibility of other biological parameters.
Our tests showed that it is possible to achieve a wide range of reproducible concentrations by modifying the bubblingairflow. These values allow us to experimentally reproduce many of the exposure levels measured in workplaces.
Other advantages of this generator are its flexibility of use and the many adjustable operating parameters it offers.Conditions can be adapted to each experiment by modifying influential parameters such as bubbling and ascending airflowrates, liquid film height, characteristics of the porous disc, concentration and composition of the microbial suspension.This modified bubbling generator provides performance levels compatible with production of bioaerosols (E. coli andendotoxins) in a large number of laboratory assays with diverse aims and constraints.
In the future, the flexibility of this generator will also be used to evaluate its performance in the production ofcontrolled bioaerosols of interest in bioaerosol science and occupational health: gram positive bacteria, actinomycetesspores or fungal spores.
X. Simon et al. / Journal of Aerosol Science 42 (2011) 517–531530
Acknowledgements
The authors would like to thank Mr. Richard Wrobel for his contribution to the size distribution measurements with theCoulters Multisizer 3 and Mr. Peter Gorner and Mr. Sebastien Bau for their useful comments throughout the study.
References
AFNOR. (1996). Microbiology of Food and Animal Feeding Stuffs—General Rules for Microbiological Examinations. NF ISO 7218, 47pp.Allen, T. (1981). Particle Size Measurement (3rd ed.). Chapman and Hall: London.Blanchard, D.C., & Syzdek, L.D. (1988). Film drops production as a function of bubble size. Journal of Geophysical Research 93, 3649–3654.Brenner, D.J. (1984). Section 5. Facultatively anaerobic gram-negative rods—Family I. Enterobacteriaceae. In: N.R. Krieg, & J.G. Holt (Eds.), Bergey’s Manual
of Systematic Bacteriology (Vol. 1). The Williams & Wilkins Co, p. 408.Brockmann, J.E. (2001). Sampling and transport of aerosols. In: P.A. Baron, & K. Willeke (Eds.), Aerosol Measurement, Principles, Techniques and Applications
(2nd ed.). Wiley Interscience: New York, pp. 143–195.CEN. (2003). (European Committee for Standardization) Atmosph�ere des lieux de travail—Determination des endotoxines en suspension dans l’air
(Workplaces atmospheres—Determination of airborne endotoxins). Norme AFNOR NF EN 14031, 13pp.Davies, C.N. (1977). Sampling aerosols with a thin-walled tube. In: Atmospheric Pollution. Elsevier: Amsterdam, pp. 433–446.Demange, M., Gendre, J.C., Herve-Bazin, B., Carton, B., & Peltier, A. (1990). Aerosol evaluation difficulties due to particle deposition on filter holder inner
walls. The Annals of Occupational Hygiene 34, 399–403.Fluent. (2006). Version 6.3.26. Fluent Inc: Lebanon, New Hampshire, USA.Georgescu, S.C., Achard, J.L., & Canot, E. (2002). Jet drops ejection in bursting gas bubble processes. European Journal of Mechanics B/Fluids,
265–280.Goyer, N., Lavoie, J., Lazure, L., & Marchand, G. (2001). Les bioaerosols en milieu de travail: guide d’evaluation, de controle et de prevention. Guide Technique.
T-23, IRSST: Montreal, 88pp.Griffiths, W.D., & DeCosemo, G.A.L. (1994). The assessment of bioaerosols: A critical review. Journal of Aerosol Science 25(8), 1425–1458.Grinshpun, S.A., Willeke, K., & Kalatoor, S. (1993). A general equation for aerosol aspiration by thin-walled sampling probes in calm and moving air.
Atmospheric Environment 27A(9), 1459–1470. Corrigendum (1994) Atmospheric Environment, 28(2), 375.Grundy, M.A., Coakley, W.T., & Clarke, D.J. (1990). Microbial content of aerosols produced from suspensions exposed to megahertz frequency ultrasound.
Ultrasonics 28(6), 415–421.Gunther, A., Walchli, S., & Rudolph von Rohr, P. (2003). Droplet production from disintegrating bubbles at water surfaces. Single vs. multiple bubbles.
International Journal of Multiphase Flow 29, 795–811.Haas, D., Unteregger, M., Habib, J., Galler, H., Marth, E., & Reinthaler, F.F. (2010). Exposure to bioaerosol from sewage systems. Water, Air and Soil Pollution
207, 49–56.Harstad, J.B., Filler, M.E., Hushen, W.T., & Decker, H.M. (1970). Homogeneous bacterial aerosols produced with a spinning-disc generator. Applied
Microbiology 20(1), 94–97.Hendricks, W., Stones, F., & Lillquist, D. (2009). On wiping the interior walls of 37-mm closed-face cassettes: An OSHA perspective. Journal of Occupational
and Environmental Hygiene 6, 732–734.Hinds, W.C. (2001). Physical and chemical changes in the particulate phase. In: P.A. Baron, & K. Willeke (Eds.), Aerosol Measurement, Principles, Techniques
and Applications (2nd ed.). Wiley Interscience: New York, pp. 83–97.Ho, J., Kournikakis, B., Gunning, A., & Fildes, J. (1988). Submicron aerosol characterization of water by a differential mobility particle sizer. Journal of
Aerosol Science 19(7), 1425–1428.Jensen, P.A., Lighthart, A.J., Mohr, A.J., & Schaffer, B.T. (1994). In: B. Lighthart, & A.J. Mohr (Eds.), Instrumentation Used with Microbial Aerosols—Theory and
Applications. Chapman and Hall: New York, pp. 226–284.Jung, J.H., Lee, C.H., Lee, J.E., Lee, J.H., Kim, S.S., & Lee, B.U. (2009). Design and characterization of a fungal bioaerosol generator using multi-orifice air jets
and a rotating substrate. Journal of Aerosol Science 40, 72–80.Juozaitis, A., Willeke, K., Grinshpun, S.A., & Donnelly, J. (1994). Impaction onto a glass slide or agar versus impingement into a liquid for the collection and
recovery of airborne microorganisms. Applied and Environmental Microbiology 60(3), 861–870.Kanaani, H., Hargreaves, M., Ristovski, Z., & Morawska, L. (2009). Fungal spore fragmentation as a function of airflow rates and fungal generation methods.
Atmospheric Environment, 1–11.Kanaani, H., Hargreaves, M., Smith, J., Ristovski, Z., Agranovski, V., & Morawska, L. (2008). Performances of UVAPS with respect to detection of airborne
fungi. Journal of Aerosol Science 39, 175–189.Kim, K., Kim, W., Yun, S.H., Lee, J.H., Kim, S., & Lee, B.U. (2008). Use of an electrospray for the generation of bacterial aerosols. Journal of Aerosol Science 39,
365–372.Krames, J., Buttner, H., & Ebert, F. (1991). Submicron particle generation by evaporation of water droplets. Journal of Aerosol Science 22(S1), S15–S18.Kubitschek, H.E. (1990). Cell volume increase in Escherichia coli after shifts to richer media. Journal of Bacteriology 172(1), 94–101.Laitinen, S.K., Kangas, J., Husman, K., & Susitaival, P. (2001). Evaluation of exposure to airborne bacterial endotoxins and peptidoglycans in selected work
environments. Annals of Agricultural and Environmental Medicine 8(2), 213–219.Lee, J.H., Hwang, G.B., Jung, J.H., Lee, D.H., & Lee, B.U. (2010). Generation characteristics of fungal spore and fragment bioaerosols by airflow control over
fungal cultures. Journal of Aerosol Science 41, 2010.Lee, B.U., Kim, S.H., & Kim, S.S. (2002). Hygroscopic growth of E. coli and B. subtilis bioaerosols. Journal of Aerosol Science 33, 1721–1723.Li, C.-S., Hao, M.-L., Lin, W.-H., Chang, C.-W., & Wang, C.-S. (1999). Evaluation of microbial samplers for bacterial microorganisms. Aerosol Science and
Technology 30(2), 100–108.Lines, R.W. (1991). The electrical sensing zone method (the Coulter principle). In: N.G. Stanley-Wood, & R.W. Lines (Eds.), Particle Size Analysis. Royal
Society of Chemistry: Cambridge, pp. 351–373.Macher, J.M. (1997). Evaluation of bioaerosol sampler performance. Applied Occupational and Environmental Hygiene 12(11), 730–736.Macher, J., Chen, B., & Rao, C. (2008). Chamber evaluation of a personal bioaerosol cyclone sampler. Journal of Occupational and Environmental Hygiene 5,
702–712.Mainelis, G., Berry, D., Reoun An, H., Yao, M., DeVoe, K., Fennell, D.E., & Jaeger, R. (2005). Design and performance of a single-pass bubbling bioaerosol
generator. Atmospheric Environment 39(19), 3521–3533.Mainelis, G., Willeke, K., Baron, P., Reponen, T., Grinshpun, S., Gorny, R., & Trakumas, S. (2001). Electrical charges on airborne microorganisms. Journal of
Aerosol Science 32(9), 1087–1110.Marchand, G., Lalonde, M., Beaudet, Y., Boivin, G., Villeneuve, S., & Pepin, C. (2007). Documentation of the endotoxins present in the ambient air of cotton
fiber textile mills in Quebec. Journal of Environmental Monitoring 9, 869–876.Palleroni, N.J. (1984). Section 4. Gram-negative aerobic rods and cooci—Family I. Pseudomonadaceae. In: N.R. Krieg, & J.G. Holt (Eds.), Bergey’s Manual of
Systematic Bacteriology (Vol. 1). The Williams & Wilkins Co, p. 165.Qian, Y., Willeke, K., Ulevicius, V., Grinshpun, A.S., & Donnelly, J. (1995). Dynamic size spectrometry of airborne microorganisms: Laboratory evaluation
and calibration. Atmospheric Environment 29(10), 1123–1129.
X. Simon et al. / Journal of Aerosol Science 42 (2011) 517–531 531
Reponen, T., Willeke, K., Ulevicius, V., Grinshpun, S.A., & Donnelly, J. (1997). Techniques for dispersion of microorganisms into air. Aerosol Science andTechnology 27(3), 405–421.
Rinsoz, T., Duquenne, P., Greff-Mirguet, G., & Oppliger, A. (2008). Application of real-time PCR for total airborne bacterial assessment: Comparison withepifluorescence microscopy and culture-dependent methods. Atmospheric Environment 42(28), 6767–6774.
Rule, A.M., Schwab, K.J., Kesavan, J., & Buckley, T.J. (2009). Assessment of bioaerosol generation and sampling efficiency based on Pantoea agglomerans.Aerosol Science and Technology 43(6), 620–628.
Scheermeyer, E., & Agranovski, I.E. (2009). Design and evaluation of a new device for fungal spore aerosolization for laboratory applications. Journal ofAerosol Science 40, 879–889.
Simon, X., Duquenne, P., & Jeandidier, V. (2009). French patent pending no. 09 59364. Cellule de generation d’aerosols et generateur d’aerosols pourvud’une telle cellule (aerosol bubbling system based on a controlled-height liquid film and aerosol generator provided with such a bubbling system).20pp.
Sivasubramani, S.K., Niemeier, R.T., Reponen, T., & Grinshpun, S.A. (2004). Fungal spore source strength tester: Laboratory evaluation of a new concept.Science of the Total Environment 329, 75–86.
Spaan, S., Schinkel, J., Wouters, I.M., Preller, L., Tielemans, E., & Tjoe Nij, E. (2008). Variability in endotoxin exposure levels and consequences for exposureassessment. The Annals of Occupational Hygiene 52(5), 303–316.
Stewart, I.W., Leaver, G., & Futter, S.J. (1997). The enumeration of aerosolised Saccharomyces cerevisiae using bioluminescent assay of total adenylates.Journal of Aerosol Science 28(3), 511–523.
Stone, R.C., & Johnson, D.L. (2002). A note on the effect of nebulization time and pressure on the culturability of Bacillus subtilis and Pseudomonasfluorescens. Aerosol Science and Technology 36(5), 536–539.
Su, W.C., & Vincent, J.H. (2004). Towards a general semi-empirical model for the aspiration efficiencies of aerosol samplers in perfectly calm air. Journal ofAerosol Science 35, 1119–1134.
Terzieva, S., Donnelly, J., Ulevicius, V., Grinshpun, A.S., Willeke, K., Stelma, G., & Brenner, K. (1996). Comparison of methods for detection and enumerationof airborne microorganisms collected by liquid impingement. Applied and Environmental Microbiology 62, 2264–2272.
Theunissen, H.J.S., Lemmens-den Toom, N.A., Burggraaf, A., Stolz, E., & Michel, M.F. (1993). Influence of temperature and relative humidity on the survivalof Chlamydia pneumoniae in aerosols. Applied and Environmental Microbiology 59(8), 2589–2593.
Ulevicius, V., Willeke, K., Grinshpun, A.S., Donnelly, J., Lin, J., & Mainelis, G. (1997). Aerosolization of particle from a bubbling liquid: Characteristics andgenerator development. Aerosol Science and Technology 26(2), 175–190.
Walter, M.V., Marthi, B., Fieland, V.P., & Ganio, L.M. (1990). Effect of aerosolization on subsequent bacterial survival. Applied and EnvironmentalMicrobiology 56(11), 3468–3472.
Wang, Z., Reponen, T., Grinshpun, A.S., Gorny, L.R., & Willeke, K. (2001). Effect of sampling time and air humidity on the bioefficiency of filter samplers forbioaerosol collection. Journal of Aerosol Science 32(5), 661–674.
