Atmospheric simulation chambers are exploratory platforms used to study
various atmospheric processes at realistic but controlled conditions. We
describe here a new facility specifically designed for the research on
atmospheric bio-aerosol as well as the protocols to produce, inject, expose
and collect bio-aerosols. ChAMBRe (Chamber for Aerosol
Modelling and Bio-aerosol Research) is
installed at the Physics Department of the University of Genoa, Italy, and it
is a node of the EUROCHAMP-2020 consortium. The chamber is made of stainless
steel with a total volume of about 2.2 m
The biological component of atmospheric aerosol (bio-aerosol) is a relevant
subject of both atmospheric science and biology. From the pioneering
investigations at the end of the 19th century (Pasteur, 1862), the
study of primary biological aerosol particles (PBAP) has definitively become
a multidisciplinary field of research, which requires expertise in physics,
chemistry, biology, and medical sciences (Desprès et al., 2012). Among
PBAP, bacteria have a crucial role (Bowers et al., 2010). They show
atmospheric concentrations from 10
So far, PBAP have been studied in-field through a variety of sampling and analysis techniques and addressing their physical, chemical, and biological properties (Reponen et al., 1995; Li and Lin, 1999; Brodie et al., 2007; Georgakopoulos et al., 2009; Fahlgren et al., 2011; Lee et al., 2010; Urbano et al., 2011). The connection between PBAP and dust dispersion and transport over very long distances (Goudie and Middleton, 2006) deserves a particular mention. Dust clouds may contain high concentrations of microbiota, e.g. fungal spores, plant pollen, algae and bacteria. Bio-aerosols associated with dust events can spread pathogens over long distances (Prospero et al., 2005; Griffin, 2007; Nava et al., 2012; Van Leuken et al., 2016) and can impact ecosystem equilibria, human health and yield of agricultural products. For many microorganisms long-range and high-altitude transport in the free atmosphere can be very stressful due to strong ultraviolet radiation, low humidity (inducing desiccation), too low or too high temperatures, and complex atmospheric chemistry (e.g. presence of radicals or other reactive species) (Després et al., 2012; Zhao et al., 2014). Only very resistant organisms are able to survive, so the composition of microbiota can change during the long airborne transport prior to deposition (Meola et al., 2015).
Airborne bacterial communities are highly diverse, and variations in their
species diversity are quite complex. The bacterial composition in air is
strongly dependent on many factors such as seasonality, meteorological
factors, anthropogenic influence, variability of bacterial sources and many
other variables. Still, the general trend from available reports is that
bacteria found in the air often belong to groups that are also common soil
bacteria (e.g. Firmicutes, Proteobacteria, Actinobacteria) (Després et
al., 2012). Due to their small size, bacteria have a relatively long
atmospheric residence time (on the order of several days or more) compared
to larger particles and can be transported over long distances (up to
thousands of kilometres). Measurements show that mean concentrations in
ambient air can be greater than
Bio-aerosols also seem to play an important role in the reactivity of particulate matter. They can induce reactive oxygen species (ROSs) production and modify particulate matter (PM) toxicity due to their ability to modulate the oxidative potential (OP) of toxic chemicals present in PM (Samake et al., 2017).
Therefore, within the bacterial survival studies there are several interconnected topics. One is related to health issues: exposure to bio-aerosols has been linked to various health effects (disease spreading, e.g. meningitis and bio-aero-contamination, like legionella and refrigerating towers. Pearson et al., 2015; Ghosh et al., 2015; Sala Ferré et al., 2009). Another topic is connected to climate and CCN and IN impact, where viability and proliferation of airborne bacteria are the significant investigation subjects (Bauer et al., 2003; Deguillaume et al., 2008; Amato et al., 2015). A biogeochemical issue is related to the long range transport of bacteria and dust events, since bacteria can stick to dust particles and can be more efficiently (i.e. remaining viable) transported over long distances. (Meola et al., 2015; Nava et al., 2012; Van Leuken et al., 2016).
The study of relevant processes taking place in the Earth atmosphere is
usually pursued through a wide range of field observations where complicated,
unexpected and interconnected effects are often difficult to disentangle. The
possibility of planning and performing experiments in controlled conditions
is therefore highly desirable. This need has triggered the conception and
development of the atmospheric simulation chambers (ASCs in the following),
i.e., small- to large-scale facilities (with volumes ranging between a few to
hundreds cubic metres), where atmospheric conditions can be maintained and
monitored in real time for periods long enough to mimic the realistic
environments and to study interactions among their constituents
(Finlayson-Pitts and Pitts, 2000). ASCs have been used to study chemical and
photochemical processes that occur in the atmosphere, such as ozone formation
(Carter et al., 2005 and references therein) and cloud chemistry (Wagner et
al., 2006) or aerosol–cloud interaction (Benz et al., 2005), but the high
versatility of these facilities allows for a wider application covering all
fields of atmospheric aerosol science. A full list and review of the approach
and of the main facilities around the world can be found in Becker (2006). In
Europe, there are several ASCs organized through the network EUROCHAMP-2020
(see all the details at the link
Since the interplay of bio-aerosol and atmospheric conditions is still poorly known, suitable facilities are needed, where transdisciplinary studies gathering atmospheric physics, chemistry, and biology issues are possible.
Experiments conducted inside confined artificial environments where physical and chemical conditions and/or compositions can be controlled, can provide information on bacterial viability, biofilm and spore formation, and endotoxin production. Currently, the literature reports several examples of studies performed in small reactors (Levin et al., 1997; Griffiths et al., 2001; Ho et al., 2001; Ribeiro et al., 2013; Sousa et al., 2012). The use of atmospheric simulation chambers has been much more limited and focused on the interaction of bacteria with atmospheric parameters, regarding bio-aerosol release effects (Jones and Harrison, 2004), and on ice nucleation and cloud condensation (Möhler et al., 2008; Bundke et al., 2010; Chou, 2011).
In 2014, some of the co-authors of the present work designed and performed an
exploratory experiment (Brotto et al., 2015) at the CESAM (French acronym
for the Experimental Multiphasic Atmospheric Simulation
Chamber; Wang et al., 2011). On
colonies of
Prompted by the outcomes of pilot experiments (Amato et al., 2015; Brotto et al., 2015), a new dedicated atmospheric chamber, ChAMBRe (Chamber for Aerosol Modelling and Bio-aerosol Research), has been designed and installed in Genoa (Italy). While ChAMBRe, as other ASCs, is a multipurpose facility, the outcomes of the correlation between bacteria viability and atmospheric condition and/or composition will provide the input for developing ad hoc modules to be then implemented in chemical transport models. This can be done following a scheme often used for the chemical mechanisms parameterization (see, for example, the smog chamber experiments used for the evaluation of carbon bond mechanisms in Parikh et al., 2013). Such software tools, are widely used both in scientific research and in air quality evaluations, to predict the fate (i.e. transport, deposition, and chemical changes) of the atmospheric pollutants and, at the moment, they do not include any biological patch.
ChAMBRe is installed at the ground floor of the building hosting the
Department of Physics of the University of Genoa, where it is jointly managed
by the Italian National Institute of Nuclear Physics (INFN) and the Physics
Department (
ChAMBRe layout.
CHAMBRe has a cylindrical shape with domed bases (Fig. 1). It has maximum
height and diameter of 2.9 and 1 m, respectively, and a total volume of
2.23 m
While ChAMBRe has been designed to operate at atmospheric pressure, the
second ISO-K250 flange of the lower cylinder is connected to a composite
pumping system (a rotary pump model TRIVAC® D65B, Leybold
Vacuum, followed by a root pump model RUVAC WAU 251, Leybold Vacuum), which
can evacuate the internal volume to a vacuum level of about
To favour the mixing of the gas and aerosol species in the reactor a fan is installed in the bottom part of the chamber (Fig. 1). It is a standard venting system with four metallic arms of 25 cm length each connected to an external engine through a rotating shaft. A particular pass through has been designed and built at INFN-Genoa to ensure the vacuum seal. The fan speed can be regulated by an external controller and varied between 0.0 and 50 Hz in steps of 0.1 Hz (0 to 3000 rpm, in steps of 6 rpm).
A set of two pressure gauges is used to measure the atmospheric pressure
inside and outside the chamber. A MKS Instruments 910
DualTrans™ transducer is installed inside with
a measuring range of
Internal temperature and relative humidity are continuously measured by a
HMT334 Vaisala® Humicap® humidity and temperature transmitter for high pressure and vacuum
application (up to 100 bars). This sensor is mounted in the upper ISO-K100
flange on the top dome. In the operative range (from 15 to 25
All the atmospheric gauges are connected to a NI Compact-RIO acquisition system (based on the NI cRIO-9064 controller), which also allows the remote monitoring of the ChAMBRe parameters through an ethernet connection.
Two types of UV lamps are permanently installed inside the chamber. A 90 cm long lamp is inserted through the flange in the top dome (Fig. 1): it
produces a 85 W UV radiation at
The large number of free flanges in the main structure gives the possibility of connecting several external instruments to ChAMBRE.
Polydispersed aerosol can be sprayed into the simulation chamber using a
Blaustein Atomizer (BLAM, single-jet model, CH Technologies), connected to
the chamber with a curved stainless-steel tube (length
Aerosol samplers and multistage cascade impactors can be easily connected
through the ISO-K flanges and maintained in operation for times depending on
their nominal flow and the needs of the particular experiment (e.g. a typical
10 L min
Particle concentration inside the chamber is measured continuously by two different instruments: a Scanning Mobility Particle Sizer (SMPS, GRIMM Technologies, Inc.) and an Optical Particle Counter (OPC, mod. Envirocheck 1.107, GRIMM Technologies, Inc.).
The SMPS is formed by three components in sequence: a neutralizer (i.e. a
bipolar diffusion charger) supplied by Eckert & Ziegler Cesio (Prague), a
differential mobility analyzer (DMA, model 55-U) and a condensation particle
counter (CPC, model 5403), both from Grimm GmbH (Ainring, Germany). The
neutralizer is based on a radioactive source of
The OPC is a Grimm 1.107 – Envirocheck version, which operates in 31 size
intervals with diameters in the 0.25–32
The ozone concentration is monitored by a M400A Ozone Analyzer from API
(Advanced Pollution Instrumentation, Inc.). The M400A uses a system based on
the Lambert–Beer law for measuring ozone in ambient air. A 254 nm UV light
signal is passed through the sample cell where it is absorbed in proportion
to the amount of the ozone present. Periodically, a switching valve
alternates measurement between the sample stream and a sample that has been
scrubbed of ozone. The instrument has a sampling rate of 0.8 L min
The nitrogen oxides (NO and
Depending on kinetics, processes in the atmosphere have typical reaction times ranging from a few seconds up to several days. For this reason, in the case of simulation chambers, the evaluation of aerosol particle lifetime is of primary importance: it is necessary to keep in suspension enough aerosol for a sufficient time, in order to allow chemical or biological transformations of particles. Aerosol particle lifetime in chambers depends on many factors, e.g. wall losses caused by adsorption or deposition, diffusion and mixing processes, gravitational settling, electrostatic drawing, or all of them combined, depending of course on particle properties (i.e. density, dimensions, shape, and vapour pressure).
Particle loss coefficient (
For the characterization of particle lifetime in ChAMBRe, the Blaustein
Atomizer (BLAM) was used. By feeding the BLAM with saline solutions (NaCl and
The presence of walls obviously influences the chemical and physical dynamics of the experiments carried out inside simulation chambers, as the gaseous species can be lost to the chamber walls. To describe the behaviour of the walls of our chamber, we considered the dark reactivity of ozone, due to its chemical reactivity towards surfaces, its relevance to chamber experiments (as reactant or as sterilization agent) and as atmospheric oxidant.
A series of five experiments have been done with initial concentration
ranging from 300 to 1000 ppbv. The ozone concentration in the chamber was
monitored as a function of time. The pseudo-first-order rate for loss
processes is equal to
The background level of particles inside the chamber was measured by SMPS
and OPC. The coupling of the two counters provides a comprehensive picture
of the particles inside the chamber ranging from few nm up to 31 microns
(for more information; see Sect. 2.3). After each experiment, the chamber
is cleaned by a multistep procedure: the UV lamp (see Sect. 2.1) is first
switched on for 10 min, the chamber is then evacuated and vented to
atmospheric pressure through an HEPA filter (Sect. 2.1). Afterwards, a
high ozone concentration (
Background level measurements performed subsequently to chamber cleaning
showed no significant particles presence (i.e. about 2 and 0.5 particle cm
Typical grow curve for
Background concentrations of
The usefulness of ASCs in providing new possibilities for the study of bacteria and other biological particles in air critically depends on the associated protocols, which are essential to understand how the bacteria survive and if they are in able to grow and reproduce in the atmospheric conditions of the simulation chamber. In this section we describe the standard methodology developed for the bio-aerosol experiments (injection, collection, and storage) and the related experimental conditions, that should be representative of the typical environmental ones.
Experimental procedures involved two strains consisting of
Correlation curve between the number of
Several techniques for bacteria and bio-aerosol characterization are available on site. In the same building that hosts the atmospheric simulation chamber there is a basic microbiology lab equipment allowing for culture analysis in vitro (isolation, identification, growth) and biochemical tests (e.g. catalase and oxidase): autoclave (Asal mod.760), vortex, centrifuge and microcentrifuge (Eppendorf centrifuge 5417R), water purification system Milli-Q (Millipore-Elix), incubator for temperature control Ecocell and Friocell MMM Group, Steril-VBH Compact “microbiological safety” cabinet, Thermo electron corporation steri-cycle HEPA Class 100 incubator; optical microscope (Nikon Eclipse TE300) for bacterial detection and live–dead discrimination by epifluorescence with specific dyes and for immunoassay fluorescence to label the antigenic bacterial target, fluorescent molecule or enzyme. The transfer of bacteria from the biological laboratory to the simulation chamber takes only a few minutes, ensuring a quickly execution of the chamber experiments, once the desired phase of bacteria growth is reached, and then a quick treatment of the samples collected after the experiments in the chamber.
The same culture preparation technique was applied at both the bacterial strains, in order to minimize experimental variations.
Correlation curve of the average count on the four Petri dishes
exposed in each experiment with the number of
Environmental parameters (RH,
Bacteria concentration (
Firstly, it is important to ensure the maximum bacteria cells viability
prior to the injection. Typically, to understand and define the growth of a
particular microbial isolate, cells are placed in a culture medium in which
the nutrients and environmental conditions are controlled. If the medium
provides all nutrients required for growth and environmental parameters are
optimal, a growth curve can be obtained by measuring the increase in
bacterial number or mass as a function of time. Different distinct growth
phases can be observed within a growth curve: these include the lag phase,
the log phase, the stationary phase, and the death phase. Each of these
phases represents a distinct period of growth that is associated with
typical physiological changes in the cell culture. Therefore, the growth
curve for both of bacterial strains was obtained quantifying the rate of
change in the number of cells in a culture per unit time thus identifying
the mid-exponential phase (log phase), where the maximum viability of the
cells is ensured and the number of dead microorganisms is at a minimum.
Before each injection we followed the bacterial growth up to the mid-exponential phase, reached in about 4 h, thus allowing the bacteria to enter the exponential phase of growth.
Spectrophotometer measurements were used to achieve the correct dilution and also to provide the first evaluation of bacterial concentration in the solution, which has to be nebulized, as explained below. The suspension was then centrifuged at 3000 rpm for 10 min, the supernatant was discarded and the pellet was evenly vortexed for 1 min in physiological solution (NaCl 0.9 %) before the injection. The cultivable cell concentration was determined following the above-mentioned procedure. The average on CFU counting is used to estimate the uncertainty range of the bacterial concentration in the nebulized solution.
In each experiment, a volume of 10 mL of the cells suspension, with a
concentration of approximately 10
Detail of
In each experiment, a volume of about 2 or 3 mL of the cells suspension was sprayed into the simulation chamber using the Blaustein Atomizer (BLAM), described in Sect. 2.3.
The main body of ChAMBRe is connected through a ISO-KF250 pneumatic valve to
a cylindrical horizontal volume which is accessible from a second ISO-KF250
gate valve (see Figs. 1 and 2). The two gate valves completely separate
the cylinder, which can be connected to the main chamber or alternatively
opened without perturbing the ChAMBRe atmosphere. This home-made device has
been specifically developed to ensure the insertion and extraction of
bio-aerosol samplers, in order to minimize the risk of contamination. This
volume can be evacuated through a bypass to the ChAMBRe main pumping system
and can be then refilled to atmospheric pressure both with particle free dry
air or through a pipe connected to the ChAMBRe main body. Inside the
cylinder, there is a sliding tray which can be inserted in ChAMBRe by a
home-made external manual control (Fig. 2) The tray can host up to six
Petri dishes (diameter 10 cm, each) which can be inserted in ChAMBRe to
collect bacteria (or in general BPAP) directly by deposition onto a proper
culture medium. The procedure to insert the Petri dishes in ChAMBRe is
organized in consecutive steps (reference to Fig. 1 for the valves names).
With V1 closed, the V2 valve is opened to allow the positioning of the Petri
dishes (pre-filled with a suitable amount of culture medium) on the sliding
tray. Valve V2 is closed and the volume inside the pipe is flushed with clean air
coming from the chamber. The atmospheric pressure inside the pipe is recovered by opening the
connection to ChAMBRe. V1 is opened and the sliding tray is completely inserted in ChAMBRe. The sterilizing UV lamp (ozone free; see section 2.2) is switched on for 15 min to guarantee the Petri dishes
sterilization. The UV lamp is switched off and ChAMBRe is ready for injection of bacteria.
The chamber sterility before the injection of bacteria was tested through a
blank experiment by injecting only sterile physiological solution: no
bacterial contamination was observed in the four Petri dishes positioned on
the sliding tray.
In a standard experiment, once the bacteria have been injected into ChAMBRe,
the Petri dishes remain exposed for the desired time and then the sliding
tray can be moved back to the pipe. The ventilation system is on during the
exposure period, to maintain a homogeneous distribution of particles inside
the chamber volume. Closing V1 and opening V2 the Petri dishes can be
removed without perturbing the conditions inside the main chamber. The
gravitational settling method has been developed to minimize microbial
damage, and has been previously proven to be a very suitable way to collect
and count viable bacteria colonies (Brotto et al., 2015). After exposure to
the chamber atmosphere, Petri dishes are incubated for 24 h at 37
Detail of
Lee et al. (2002) suggest that the average aerodynamic diameters of generated
Environmental parameters (RH,
Bacteria concentration (
Bacteria from the original liquid suspensions, both in broth and in
physiological solution (Sect. 4.2), were also collected on polycarbonate
filters (Isopore membrane track-etched filters, pore size 0.05
Experiments to study the correlation between bacterial viability and the atmospheric composition and conditions in ChAMBRe rely on an assessed protocol to inject and extract bacteria from the chamber. A first set of experiments was therefore devoted to measuring the reproducibility of the whole process with a clean atmosphere (i.e. with the background levels given in Sect. 3.3) inside ChAMBRe.
Five different experiments were performed in the period from July and
November 2017. The protocol described in Sect. 4 was followed for the
bacteria growth, the injection in the chamber and the bacteria collection by
four Petri dishes inserted by the sliding tray (Sect. 4.3). Values of the
atmospheric parameters in ChAMBRe during each experiment are reported in
Table 1. The bacteria concentrations measured in the aerosolized solution and
the average number of colonies counted on the Petri dishes after the exposure
in ChAMBRe are reported in Table 2. The volume of the bacterial suspension
injected through the BLAM atomizer was equal to 2 mL, except during the
fourth experiment where the volume was increased to 3 mL (Table 2). This
ensured that the concentration of viable bacteria injected in the chamber was
comparable to the values typical of the real atmosphere (Bauer et al., 2003;
Burrows et al., 2009b). Taking into account the BLAM nebulization efficiency
(Sect. 4.2), the initial aerosol concentration of living microorganisms in
ChAMBRe after the injection, was estimated to be around
10
Five different experiments were performed in the period from January and
March 2018, following the protocol described in Sect. 4. The values of the
atmospheric parameters in ChAMBRe are reported in Table 3. In this set of
experiments the relative humidity inside the chamber was increased up to 70 %, compared to the environmental value recorded in the laboratory, by
changing the working condition of the humidifier (Benbough, 1967; Cox, 1966;
Dunklin and Puck, 1947).
The dilution factor, the bacterial concentrations measured in the
aerosolized solutions, and the average number of colonies counted on the
Petri dishes after the exposure in ChAMBRe are reported in Table 4. It is
worth noting that in the experiments discussed in Sect. 5.1, a narrow
interval of OD
The volume of the bacterial suspension injected through the BLAM atomizer was
equal to 2 mL in the first four experiments and was increased to 2.8 mL in
the fifth experiment (Table 4). Figure 6 shows the correlation between the
number of injected and collected CFU (top panel), indicating that the
uncertainty on the slope of the correlation curve (about 4 %) was even
better than the same uncertainty related to
Although for this bacterial strain a less concentrated solution was injected, more CFUs were collected on the Petri dishes placed inside the chamber. This result could depend on the fact that the humidity in the chamber was generally greater in the second set of experiments providing Gram-negative microorganisms with a more comfortable environment, but also it could depend on the behaviour of the two different bacteria strains.
The FESEM micrographs (Figs. 7 and 8) of the bacteria contained in the
liquid suspensions before injection (see Sect. 4.3) clearly show that the
cells of
A new atmospheric simulation chamber, ChAMBRe, has been installed at INFN-Genoa. The facility has been designed to perform experimental studies on primary biological aerosol particles and bacteria in particular. The performance of the new chamber, which may impact on the future experiments on bio-aerosol (i.e. wall reactivity, aerosol particle lifetime, background levels), has been quantitatively assessed. Furthermore, a protocol to handle the injection and extraction phases has been thoroughly tested both with Gram-positive and Gram-negative bacterial strains. With a clean atmosphere maintained inside ChAMBRe, the ratio between injected and extracted viable bacteria turned out to be reproducible at a 10 % level. Such a result is the first methodological step in view of a forthcoming systematic study of the correlation between bacterial viability and pollution levels. Resident times of viable bacteria in ChAMBre are less than 5 h, much shorter than the generic residence time in the open atmosphere. However, previous literature studies (Brotto et al., 2015) suggest that such a time window is long enough to observe the effects (i.e. viability change) of bacteria exposure to air pollutants. The assessment of such effects is objective of the forthcoming studies at ChAMBRe.
The EUROCHAMP-2020 consortium has a data center where every
piece of information produced by members is stored and available to everyone.
The link is as follows:
DM, PB, FP, and PP designed and built ChAMBRE; DM, SGD, and PP ran all the injections with bacteria; SGD, EG, ADC, and LV took care of all the biological issues and measurements; AC, CC, LN, and MO performed the SMPS measurements and the FESEM analyses; DM, SGD, CC, MO, JFD, PF, and PP performed the measurements to assess the aerosol lifetime in ChAMBRe and the wall reactivity; FF designed and implemented the acquisition software; JFD and PF provided advice from their long-standing expertise in the field; DM, SGD, CC, EG, and PP prepared the article with contributions from all of the other authors.
The authors declare that they have no conflict of interest.
This project has received funding from the European Union's Horizon 2020 research and innovation programme through the EUROCHAMP-2020 Infrastructure Activity under grant agreement no. 730997. The Authors are indebted to the technical staff of INFN-Genoa for the intense and talented electro-mechanical work. Jean François Doussin wishes to thank the Physics department of the University of Genoa for granting scientific invitations that have allowed its participation to this work. The authors wish to thank Houssni Lamkadam and Claudia Di Biagio (LISA) for the Lai and Nazaroff calculations. Edited by: Johannes Schneider Reviewed by: two anonymous referees