Natural sources and experimental generation of bioaerosols: Challenges and perspectives

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Citation Alsved, Malin et al. “Natural Sources and Experimental Generation of Bioaerosols: Challenges and Perspectives.” Aerosol Science and Technology 54, 5 (May 3, 2020): 547–71.

As Published http://dx.doi.org/10.1080/02786826.2019.1682509

Publisher Informa UK Limited

Version Author's final manuscript

Citable link https://hdl.handle.net/1721.1/125425

Terms of Use Creative Commons Attribution-Noncommercial-Share Alike

Detailed Terms http://creativecommons.org/licenses/by-nc-sa/4.0/

Natural sources and experimental generation of bioaerosols: Challenges and Perspectives

Malin Alsved1, Lydia Bourouiba2, Caroline Duchaine3, Jakob Löndahl1, Linsey C. Marr4, Simon T. Parker5, Aaron J. Prussin II4, Richard J. Thomas5*

1Ergonomics and Aerosol Technology, Department of Design Sciences, Lund University, Lund, Sweden

2The Fluid Dynamics of Disease Transmission Laboratory, Massachusetts Institute of Technology, Cambridge, MA 02139, USA

3Dèpartement de biochimie, de microbiologie et de bio-informatique, Facultè des sciences et de gènie, Universitiè Laval, Quèbec City, QC, Canada

4Civil and Environmental Engineering, Virginia Tech, Blacksburg, VA 24061, USA

5Defence Science & Technology Laboratory, Porton Down, Salisbury, UK

*CONTACT Richard J. Thomas [email protected] Defence Science & Technology Laboratory, Porton Down, Salisbury, UK.

Received: 6 July 2019; Revised: 5 October 2019; Accepted: 8 October 2019

Abstract

Experimental aerosol generation methods aim to represent natural processes; however the complexity is not always captured and unforeseen variability may be introduced into the data. The current practices for natural and experimental aerosol generation techniques are here reviewed. Recommendations for best practice are presented, and include characterization of starting material and spray fluid, rational selection of appropriate aerosol generators, and physical and biological characterization of the output aerosol. Reporting of bioaerosol research should capture sufficient detail to aid data interpretation, reduce variation, and facilitate comparison between research laboratories. Finally, future directions and challenges in bioaerosol generation are discussed.

1. Introduction

Bioaerosols are airborne entities that either contain microorganisms or biological materials derived from living organismsAccepted, mixed with solids or fluids (Depres Manuscript et al., 2012). When referring to bioaerosols, it is important to distinguish between two primary types: dry (particles) and liquid bioaerosols (droplets). The latter can remain in liquid phase, or evolve into dry particles depending on their local environment (Lighthart, 1994). The nature of the dried bioaerosols resulting from evaporation of droplets is distinct from that of bioaerosols originally formed as dry particles (Cox, 1970; Cox, 1987). Understanding the nature and impact of bioaerosols requires recognition of interactions between several aspects of the aerosol system, such as (i) source of aerosolized material, (ii) aerosol generation method, (iii) atmospheric transport/processing of bioaerosol particles, (iv) aerosol sampling/deposition, and (v) down-stream quantification techniques. This review focuses on the initial two aspects: sources and mechanisms of generation of bioaerosols. The combination of source and mechanism defines the process of aerosol generation, and the aim in experimental aerosol generation studies should be to mimic the natural process. For example, liquid aerosol generation defines the initial droplet which is the micro-environment where biological components reside (Haddrell & Thomas, 2017).

In this review natural sources and mechanisms of bioaerosol generation in the environment are described with respect to experimental techniques. Recommendations are made regarding experimental design, future direction and reporting of experimental methods with the aim to aid interpretation of data, and reproducibility between laboratories undertaking bioaerosol research. In order to fully understand and standardize reporting of bioaerosol data the entire aerosol system from generation of droplets to sampling and quantification of the biological components requires consideration. In view, readers are directed to reviews in this special issue covering Bioaerosol Research: Methods, Challenges and Perspectives.

2. Natural sources and generation of bioaerosol

Bioaerosols ejected into the air can be transported thousands of kilometers before deposition and are even found in cloud droplets (Morris et al., 2008). They are thus an integral part of dispersal of biological material across the globe. Natural sources of bioaerosols are diverse, and emissions of bioaerosols into the atmosphere can be segregated into two major categories based on their nature upon formation and ejection from their source: liquid (droplets) or dry (particles). They are formed via a number of mechanisms falling in two groups: natural or anthropogenic systems, including the built environment (Figure 1a). The built environment in which humans live includes buildings, parks, transportation systems, utilities and associated infrastructure such as ventilation systems (Prussin & Marr, 2015). In most cases, an individual microorganism is not launched by itself but may be accompanied by liquid, solutes, and/or abiotic solid material when dispersing short or long distances before eventually depositing. Following deposition, on surfaces, bioaerosols can also be resuspended by airflow, including from wind outdoors, or ventilation such as walking or opening doors indoors. (Bhangar et al., 2016; Inizan, 2018). Furthermore, peaks in aerosol concentrations, particle size distributions and variations in biodiversity are often observed in natural situations where they evolve and vary as a function of time and seasonality (Roses-Codinanchs et al., 1992; Burrows et al., 2009; Celenk et al., 2009; Simon & Duquenne, 2013; Lofgren et al., 2017; Caliz et al., 2018).

2.1 Dry aerosols: particles

Dry natural aerosols are produced by mechanical processes that can lift biological material from the source surface into the atmosphere by direct suspension or by saltation, as in dust storms or wind in cities (Kellogg & Griffin, 2006; Griffin & Kelly, 2004; Griffin et al., 2007; Polymenakou et al., 2007; Soleimani et al., 2016), although the detailed mechanisms behind these processes remain poorly understood, a rich interplay between fluid and mechanical properties of complex matter, fracture, and adhesion are involved (Despres et al., 2012). Dry bioaerosols can be generated from wind-induced mechanical motion, from resuspension (Langre, 2008; Inizan, 2018), and from wet/liquid mechanicalAcceptedly induced processes. An example Manuscript of the latter is the impact of raindrops on surfaces that can generate convective flows, vortices, or mechanical explosive ejections promoting efficient dispersal of pollen grains and spores (fungal, ferns) (Kim et al., 2019; Roper & Seminara, 2019; Trail et al., 2005; Niklas, 1985; McCartney, 1994; Roper et al., 2010).

Dry bioaerosols may also be generated by organisms, including humans, livestock, wildlife, and microorganisms. Humans shed on the order of 108 cells and commensal microorganisms per day, and have been shown to be a major contributor to bioaerosols by release of skin and hair fragments during movement (Milstone, 2004; Hospdsky et al., 2012; Adams et al., 2015; Qian et al., 2012; Colbeck & Whitby, 2019). Municipal waste handling contributes to substantial amounts of bioaerosols, including bacteria, fungal spores and endotoxins (Pillai et al., 2002; Folmsbee et al., 1999). During mechanical agitation in a green compost facility, a 3-log increase in airborne Aspergillus fumigatus concentrations was reported (Taha et al., 2006). Elevated bioaerosol concentrations have also been detected in the air around landfills (Huang et al., 2002; Lis et al., 2004).

2.2 Liquid aerosols: droplets

Liquid bioaerosols, emitted in the form of droplets, are ubiquitous in the environment as more than 70% of the earth is covered by water and bacteria concentrations in the ocean are >108 cells per liter (Azam et al., 2013). It is therefore likely that the majority of bioaerosols in the atmosphere originate from natural bodies of water, including both saltwater and freshwater systems. Droplet bioaerosols are produced from a range of mechanisms involving fluid fragmentation (Bourouiba & Bush 2013; Eggers & Villemaux, 2008). Woodcock and Gifford (Woodcock et al., 1949) were pioneers in showing that aerosols are generated from sea-spray (Blanchard & Syzdek, 1982; Blanchard, 1983). The largest aerosols produced from natural bodies of water are typically spume drops with diameters up to a few millimeters (Andreas, 1998; Veron, 2015).

One of the main aerosolization mechanisms from waters are bursting bubbles. Air bubbles are ubiquitous and populate the surfaces of pools, rain puddles, and wastewater treatment plants, in addition to oceans and fresh water bodies. They are produced during wave breaking, or during rainfall impacts causing splashes that generate bubbles in addition to droplets, with an estimated 1019 bubbles created every second in Earth’s oceans and seas (Poulain & Bourouiba, 2019). Bubbles produce two groups of droplets: jet drops (10 – 100 µm) and film drops (0.01 – 10 µm). Small bubbles below the capillary length produce jet drops via cavity collapse, while bubbles above the capillary length produce film drops from disintegration and destabilization of their cap (Walls et al., 2014; O'Dowd & De Leeuw, 2007; Resch et al., 1986; Walls et al., 2014; Löndahl, 2014). The produced droplets can carry biological material, salts and other organic materials (Fitzgerald, 1991; O'Dowd et al., 2004), and their load was shown to be directly affected by plankton blooms (Fitzgerald, 1991). More recently, it became clear that natural processes of generation of such droplets involve a subtle interplay between the fluid phase and the biological organisms: the latter can manipulate the underlying interfacial physics to enhance their own dispersal, via bubble bursting by producing secretions that can ultimately change by orders of magnitude, the number, sizes, speed of ejections, and compositions of the droplets launched (Poulain & Bourouiba, 2018). Similarly, changes in salinity, volatile compounds, surfactants and temperature can dramatically modify the production of droplets from bubbles, radically changing the lifetime of bubbles, which in turn, controls the number and sizes of droplets (Poulain et al., 2018; Poulain & Bourouiba, 2019). These processes highlight the importance of the interplay between the organisms and the fluid phase in shaping natural or engineered emission of bioaerosols.

Similarly to gas bubbles that burst, impacts by raindrops can also generate droplet bioaerosols of a wide range of sizes (Fitt et al., 1989; Madden, 1997 ; Joung et al., 2017). Splashes generate secondary droplets upon raindrops impacting on soil, vegetation, or animal manure (Aylor, 1990; Hau & de VallaveilleAccepted-Pope, 2006; Dungan, 2010; Milner, Manuscript 2009; Gilet & Bourouiba 2014). These splash impactions contribute to disease transmission in crop systems, in addition to dispersal of organisms broadly (Madden, 1997; Gilet & Bourouiba, 2015; Wang & Bourouiba, 2018a; Lejeune et al., 2018).

Wastewater treatment plants, especially aeration basins, have been shown to release bacteria, and viruses into the atmosphere (Wang et al., 2018; Karra & Katsivela, 2007; Sánchez-Monedero, 2008). Further, these microorganisms may carry antibiotic resistance genes, facilitating spread of resistance in the environment (Li et al., 2016). Another source are cooling towers that are notorious for being the source of bacteria during many Legionnaires’ Disease outbreaks (Fields et al., 2002; Hamilton et al., 2018; Fitzhenry et al., 2017; Gallagher, 2017). Liquid bioaerosols are also released during human hygiene practices such as showering, toilet flushing, and operating taps often due to biofilm growth on appliances and microbes present in water (Johnson et al., 2013; Barker & Jones, 2005; Zhou et al., 2007; Verani et al., 2014; Feazel et al., 2009; Thomson et al., 2013; Traverso et al., 2013), resulting in a wide range of droplet sizes (Johnson et al., 2013; Bollin et al., 1985). Finally, humans and animals produce liquid bioaerosols when breathing, coughing, sneezing, or vomiting (Fabian et al., 2008; Heo et al., 2017; Johnson & Morawska, 2009; Xie et al., 2009; Bourouiba et al., 2014; Scharfman et al., 2016; Bourouiba, 2016; Alsved et al., 2019). Exhalations are emitted in the form of a discrete high-momentum turbulent cloud, a ‘puff cloud’, that is multiphase, comprised of gaseous phase: the turbulent air exhaled; the liquid phase: the forcibly ejected respiratory tract liquid fragmenting into droplets; and eventually, the solid phase: the droplet residues remaining suspended in the turbulent cloud (Bourouiba et al., 2014; Scharfman et al., 2016; Bourouiba, 2016; Bourouiba, 2018) Here, similarly to wind-dispersal, it is not primarily the droplet characteristics that determine their range of dispersal, but the properties of the turbulent puff cloud laden with the droplets that governs the range of dispersal of the bioaerosols (Bourouiba et al., 2014; Bourouiba, 2016; Bourouiba, 2018).

3. Experimental generation of bioaerosol

Experimental aerosol generation aims to replicate aspects of natural bioaerosol generation in a controlled environment. These experiments enable greater understanding of airborne phenomena such as viability or transformation of microorganisms and biological material in the atmosphere. Biodiversity and peaks in concentration are often not considered or replicated in laboratory experiments, yet are important to the application of research to real bioaerosols, for example, biocollector efficiencies, risk assessment of microbiological or toxicological exposure and effectiveness of protective technologies against bioaerosols (Simon & Duquenne, 2013; Degois et al., 2019). Reproducible particle concentrations in laboratory experiments are typically higher than those that occur naturally in order to facilitate detection and statistical analysis. Experimental generation of bioaerosol can be considered as a number of steps, each potentially critical to variability and interpretation of data: preparation and storage of material pre-aerosolization, followed by aerosol generation and characterization. It is critical to account for the key interplay between the coupled organisms and the fluid phase in determining selection of the droplet size distributions, numbers, and compositions produced by a given fluid fragmentation process. The underlying physics of the fluid fragmentation, either impact-driven or shear-driven, steady or unsteady, involving Newtonian or non-Newtonian fluids, involves a particular relation between the properties of the mixture, fluid and organisms, and the resulting droplets generated to form the final spray.

3.1 Preparation and storage of material

Preparation, storage, and characterization of material prior to aerosolization is necessary for understanding the outcome of a study, which may vary depending upon the choice of aerosol generation device. For example, pollen and fungal spores represent dried products and therefore should be aerosolized as such, whilst bacteria and viruses are commonly found naturally in liquid suspensions and areAccepted hence aerosolized from liquids. Culture Manuscript conditions of microorganisms can influence phenotype and survival within experimental systems. Hence, aspects such as media type, cell line, temperature, humidity, pH, aeration, incubation time and final concentration must be considered as important variables between laboratory studies (Cox et al., 1971; Cox, 1987; Handley & Webster, 1995; Hogan et al., 2005; Faith et al., 2012). Storage conditions (e.g. media, temperature, humidity and time) may all influence the quality of the material to be aerosolized and introduce variability. Standard conditions of laboratory preparation may be very different from the nutrient- sparse, stressful conditions of natural environments and this may impact microbial physiology in the spray suspension and in the aerosol phase. Aggregation of particles during storage is a problem for dried material due to presence of moisture, and various inter-particle attractive forces and can influence efficiency of dispersal (Calvert et al., 2009; Masuda, 2009).

Spray fluid characteristics such as solute/solvent composition, fluid density, viscosity and surface tension alter the initial droplet size, hygroscopic growth and evaporation kinetics (Gilet & Bourouiba, 2015; Wang & Bourouiba, 2018b; Eggers & Villermaux, 2008; Johnson et al., 1999a; Yang & Marr, 2012; Haddrell & Thomas, 2017; Vejerano & Marr, 2018; Kooij et al., 2018). These characteristics, in turn, influence aerosol transport and atmospheric processing prior to sampling. For example, differences occur in survival of microorganisms when comparing experimental spray fluids to those replicating natural sources (Barlow et al., 1973; Donaldson, 1972; Trouwborst & Kuyper, 1974; Ijaz et al., 1985; Lever et al., 2000; Zuo et al., 2014). The starting concentration and aggregation of biological material within spray fluid impacts particle loading and size. This in turn affects physical deposition rates, aerosol survival and infectivity (Hogan et al., 2005; Eninger et al., 2009).

Clearly, characterizing the material to be aerosolized in terms of quality such as viability of the organism, stability of chemicals and phase, and presence of aggregations is important for reproducibility. It is recommended that sufficient material be prepared and if necessary aliquoted for the duration of a study to aid consistency. How the biological material used is prepared and stored should be documented and standardized within studies and where appropriate between laboratories. Rationales for selection of methods (including standardization) should be supported by experiments exploring variability within preparation and storage parameters. Experimentally, the physicochemical properties of spray fluid should represent the natural source in experiments aiming to model ambient phenomena. These principles should be considered for all bioaerosols with full details provided in methodology.

3.2 Laboratory liquid bioaerosol generation

3.2.1 Fluid fragmentation into droplets

Selection of an aerosol generation technique involves consideration of several aspects related to both the aerosol and the biological material being investigated. The aerosolization principle being simulated, size and polydispersity of aerosol particles, and required particle concentration are important considerations. Biologically, the quantity of material available, size and sensitivity of the biological entities, and the stresses to which the bioaerosol is exposed during generation are important (Figure 1b). These aspects are usually dictated by the natural phenomenon under research. Numerous devices for generation of bioaerosols are available (Table 1). However, many are adapted from aerosolization techniques for non-biological material and thus not optimized for bioaerosol studies.

Many natural sources of bioaerosol arise from wet environments, as described in the previous section, and these are replicated in the laboratory by fragmentation of liquids. Fragmentation is the breakup of bulk fluid into droplets which occurs when forces imposed on the system overcome surface tension forcesAccepted that tend to minimize creation ofManuscript new surface area. The Weber number (We) is the non-dimensional number that quantifies competition between kinetic energy and surface energy, defined as We = (ρv2L)/σ, linking fluid density (ρ), speed (v), length-scale (L) and surface tension (σ). When We is high, creation of new surface in the form of fragmentation of a bulk fluid into droplets is possible (Lefebvre & McDonnell, 2017; Bourouiba & Bush, 2013). Fragmentation is induced by (i) impacts, transforming a bulk fluid into a sheet, then ligaments, and then droplets via a series of surface-tension dominated interfacial instabilities and processes (Wang & Bourouiba, 2017; Wang & Bourouiba, 2018b; Eggers & Villermaux, 2008); (ii) shearing, from an airflow or one fluid moving faster over the interface of another, leading to classical hydrodynamic instabilities (i.e. Kelvin-Helmholtz), resulting in ligament, and then droplet formation (Eggers & Villermaux, 2008); (iii) or bubble bursting, leading to the creation of secondary droplets, for example from film rupture and destabilization into ligaments, and then droplets (Walls et al., 2014; Poulain & Bourouiba, 2018).

In these processes, fragmentation is influenced by both the fluid properties (Newtonian vs non- Newtonian rheology) and the regimes in which fluid destabilization occurs, such as inertial vs viscous regimes, or steady vs unsteady processes (Wang et al., 2018; Wang & Bourouiba, 2018b). A frequently used bioaerosol generator is the twin-fluid Collison nebulizer that generates droplets by physical shearing and impaction onto a vessel wall. The resultant droplets that exit the orifice of the Collison nebulizer eventually become fine dried particles of average sizes generally less than 2 µm (May, 1973). The Collison nebulizer has been used to generate larger dried particles including polystyrene latex beads and fungal spores up to around 3 µm diameter (Wang et al., 2004; Yao & Mainelis, 2006; Grinshpun et al., 2007), however particles greater than 5 µm presented problems (Kaanani et al., 2008). Benefits of the Collison nebulizer are user-friendliness, the relatively small volume of material needed, high reproducibility, high particle output (especially for the multiple-jet devices) and its widespread application, which facilitate comparison among studies (May, 1973; Liu & Lee, 1975; Reponen et al., 1997; Ibrahim et al., 2015). Several other devices use a similar principle of twin-fluid fragmentation (Table 1), however, a main disadvantage of most of these devices is the repeated recirculation of the liquid. Biological material may be damaged and/ or lose viability due to repetitive exposure to shear forces during atomization and impaction against the reservoir wall resulting in gradual degradation of the starting material in a time- and pressure- dependent manner (Stone & Johnson, 2002; Zhen et al., 2013; Turgeon et al., 2014; Zhen et al., 2014; Ibrahim et al., 2015; Haddrell & Thomas, 2017; Astudillo et al., 2018; Otero-Fernandez et al., 2019). Recirculation has been found to be most detrimental to microbes with a cell membrane, such as bacteria. Conversely, non-enveloped viruses, and bacterial or fungal spores with more rigid exteriors may be less damaged by mechanical forces (Zhen et al., 2014; Turgeon et al., 2014). Even aerosols of lipid based liposomes and microtubules demonstrate size reduction due to breakage (Niven et al., 1992; Johnson et al., 1999b). However, a recent report demonstrates the validity of comparing generation effects across viruses and bacteria. A vibrating mesh aerosol generator when compared to a 3-jet Collison nebulizer demonstrated increased viability for influenza virus, rift valley virus and encephalitic alphaviruses. This effect was not observed for a vegetative bacterium, Francisella tularensis aerosolized in the same manner (Bowling et al., 2019). It is recommended that the effect of aerosol generation be assessed for each bioaerosol and a range of generation mechanisms assessed to select the least detrimental method (Zhen et al., 2014; Turgeon et al., 2014; Astudillo et al., 2018; Bowling et al., 2019). It is also recommended that reported methodology should include details of spray system, including nozzle type, characteristic dimensions and materials of the system (including nozzle/ impaction surface), and operating parameters, including fluid composition and associated properties such as liquid and gas flow rates, pressures, timescales, and liquid volumes used. Furthermore it is recommended that studies be conducted to better understand the detail required to improve repeatability in bioaerosol studies; for example understanding the effects of fluid composition and associated properties such as static surface tension, density, rheology, and dynamical parameters such as the Weber number.

3.2.2 Replication Acceptedand validity Manuscript

Replicating natural bioaerosol formation mechanisms is not simple but does have the advantage of ruling out a potential source of variation when extrapolating laboratory generated data to natural processes. Pollen and spores often have hydrophobic surfaces, and therefore predominantly reside at liquid-air interfaces (Reponen et al., 1996) where they can be aerosolized by bubble bursting (Simon et al., 2013). Various set-ups have been constructed to replicate natural bubble bursting processes such as ‘bubble tanks’ and film drop generation (Fuentes et al., 2010; Perrott et al., 2017; Alsved et al., 2018; Joung et al., 2017; Poulain & Bourouiba, 2018).). Additionally, wind tunnels have been used to replicate wind-blown generation of bioaerosol (Taha et al., 2005). A difficulty is that these systems generally produce very low aerosol concentrations.

Replication of the bubble bursting mechanism has been achieved in the laboratory with a scaled device (Reponen et al., 1997) with further enhancement preventing recirculation of biological material (Mainelis et al., 2005) extending the range of experimentation possible in the field (Rule et al., 2009; Simon et al., 2011; Simon et al., 2013; Alsved et al., 2018). Bioaerosol generation using bubbling principles that mimic natural formation is used to study preferential aerosolization mechanisms and to compare the differential behavior of bacteria or viruses strains (Perrott et al., 2017; Gauthier et al., 2016).

Reducing stress or damage to microbes during the generation process would aid reproducibility in bioaerosol studies and can be achieved in a number of ways. Assessment of operational parameters such as spray fluid (i.e. viscosity or solute composition), air flow velocity/pressure or spray/drying time, on damage to biological materials will enable selection of appropriate conditions for a particular bioaerosol generation system (Zhen et al., 2014). Additives, such as artificial mucus or allantoic fluid from embryonated eggs (Turgeon et al., 2014) may be included in the nebulization fluid in order to reduce aerosolization stress and reproduce natural environments. Many spray devices that operate by alternative fragmentation mechanisms in a single-pass mode are now available, where the biological material only passes through the nozzle once prior to aerosolization with resultant low rates of damage to the material (Table 1). However, even gentle processes may be detrimental and each device requires an assessment against a particular biological material. A potential disadvantage of single-pass techniques is the requirement for larger volumes of valuable starting material, where only a small fraction is aerosolized. Many devices offer advantages over the Collison nebulizer in terms of expanding the initial particle size distributions closer to those represented by natural sources. For example, ultrasonic nebulization and centrifugal atomization techniques have been used to generate reproducible aerosol concentrations and distributions greater than 5 µm (Dybwad & Skogan, 2017; Bohannon et al., 2015). Piezoelectric droplet-on-demand generators offer excellent monodispersity in a highly controlled reproducible manner (Ulmke et al., 2001; Vaughn et al., 2016; Otero-Fernandez et al., 2019), that provide promise in covering the range of droplets generated from exhalation events (Xie et al., 2009).

Some studies investigate natural processes within laboratory settings, for example, measuring the size and concentration of exhaled bioaerosols generated from human respiratory activities (Fennelly et al., 2004; Wainwright et al., 2009; Xie et al., 2009; Yan et al., 2018; Scharfman et al., 2016; Bourouiba et al., 2014). Such studies provide a better physicochemical representation of the source and regime of dispersal of respiratory bioaerosols, however there may be aspects worthy of further research such as inter- and intra-personal variability (healthy versus infected, changes over disease course), and interventions that can modify bioaerosol generation (Lindsley et al., 2012; Bischoff et al., 2013; Lofgren et al., 2017; Asadi et al., 2019).

3.3 Laboratory dry bioaerosol generation and validity

In comparison to bioaerosolsAccepted from liquid, bioaerosols fromManuscript dried material are much less studied in laboratory settings. However, this mode of generation is important as bioaerosols of pollen grains and fungal/ bacterial spores are naturally generated from dry environments due to disturbances by airflows, and frequently exist as aggregates (Niklas, 1985; Lacey, 1991). Experimental techniques for dry bioaerosol generation often use an airflow pointed toward either powdered material or a sporulating fungal agar culture (Cox et al., 1970; Reponen et al., 1997; Lee et al., 2008), or alternatively using a scraping/ brushing mechanism to detach material into an air flow (Wang et al., 2014). Pollen dispersal has been reproduced in laboratory studies by taking grass or catkins from the environment into a controlled chamber and replicating the moisture-drying cycle causing release of pollen (Taylor et al., 2002; Taylor et al., 2004). There is great potential to translate techniques developed for aerosolization of non-biological particulates such as metal oxides, nanoparticles and therapeutics (Tang et al., 2008; Calvert et al., 2009; Masuda, 2009; Tsai et al., 2012; Tiwari et al., 2013). The primary experimental dispersion mechanisms for dried powders include individual or combinations of the following processes: (1) entrainment into accelerating or decelerating air flow i.e. eductor or venturi, (2) break-up of particles through impaction onto a target that may be stationary or moving i.e. fluidized bed, and (3) mechanical disruption of powdered material into air flow i.e. scraping (Table 1; Calvert et al., 2009). In addition, the dried material is required to be delivered to the dispersal mechanism, for example, by a vibrating tray or hopper, prior to being aerosolized (Calvert et al., 2009; Masuda, 2009; Pokharel et al., 2019).

In the above processes there are technical challenges that can affect data interpretation. Consideration of production and storage conditions for dried material is important as humid atmospheres may cause material to clump and compact (Inizan, 2018). Thus storage can affect the flowability of the material within feeding mechanisms and break-up during aerosolization (Gόrny et al., 2002; Masuda, 2009). Irrespective of storage conditions, particles less than 10 µm have high attractive forces between particles and to surfaces that must be overcome for efficient dispersal (Beaudoin et al., 2015), and these effects are highly sensitive to humidity (Inizan, 2018). Preparation method can affect the size of the dried particles, for example milling parameters influenced the final size of Bacillus thuringiensis particles used as a biopesticide (Kim & Je, 2012). Methods that use a feeding mechanism should have continuous stable flow of the dried material in quantities that facilitate delivery of a constant concentration of particles to the dispersal mechanism (Masuda, 2009; Pokharel et al., 2019). Problems with these factors can provide difficulties in maintenance of stable particle concentrations in the aerosol, particularly at the beginning of aerosolization, where concentration peaks may occur that subside and stabilize over time (Tang et al., 2008; Calvert et al., 2009). Generally introduction into a moving air stream with acceleration through small exits will deagglomerate loosely attached clumps as occurs in devices using the eductor and venturi principles (Masuda, 2009; Calvert et al., 2009; Tiwari et al., 2013). Issues with agglomeration and generation of particles of the correct size can be reduced by incorporation of systems downstream that remove larger particles such as impactors or cyclones prior to measurement (Pokharel et al., 2019). The development of the swirling flow disperser overcame this technical challenge for some bioaerosols, delivering stable particle concentrations for up to an hour facilitating reproducibility (Reponen et al., 1997).

3.4 Aerosol characterization

3.4.1 Liquid bioaerosols characteristics at the source

The size distribution of aerosolized biological material is vital for its transport, transformation in the air and deposition on surfaces or in a sampler. It is not always controlled by the physical size of the organism. An example is that some biological components of aerosol droplets are so small (i.e. viruses and proteins) that the resultant droplet size is initially controlled by the properties of the spray fluid and method of aerosol generation. Thereafter, the resulting droplet size distributions will evolve in a manner specific to the particular droplet fragmentation process. Thus, subtle design differences and variationAccepted in operational parameters (e.g. Manuscript pressure and viscosity), lead to variation in physical aerosol characteristics both at the source and downstream (Gussman, 1984; Hogan et al., 2005; Bourouiba & Bush, 2013; Poulain & Bourouiba, 2018; Wang & Bourouiba, 2018b; Wang et al., 2018). Physical characteristics of the aerosol should be described, including the full droplet size probability density function, the concentration of droplets generated, and for consistency with current literature, the resulting moments of the distribution, such as the mass median aerodynamic diameter (MMAD) or the geometric standard deviation (GSD). Many particle sizing technologies exist and an appraisal is outside scope of this review, other than to strongly recommend that capturing and reporting such information becomes standard across bioaerosol research communities. 3.4.2 Liquid bioaerosols characteristics: monitoring evolution from source

Liquid aerosolization initially generates wet droplets that evaporate in a time-dependent manner as a function of relative humidity, volatility and composition of the liquid, and temperature of the surrounding environment. At relative humidities below 30%, equilibrium size (dried particle) is reached within a few seconds for micrometer sized droplets. This process is important because microorganisms often lose viability rapidly within the first minutes of aerosolization (Hayakawa & Poon, 1965; Cox, 1987), presumably as the initial large droplets evaporate to reach equilibrium size (Haddrell & Thomas, 2017). Particle size distribution information is generally collected at a distance from the orifice of the aerosol generator and would be assumed to represent equilibrium particle sizes. However, without monitoring how the distribution changes over distance within the experimental apparatus this may not be a safe assumption for the reader without explicit statement within an article. Factors that impact evaporation and equilibrium particle size such as humidity, temperature and how they are controlled within the experimental system should be reported in methodology. Further it is recommended that consideration be given to how evaporation stresses can impact an experiment with bioaerosol. . Monitoring the aerosolized particles for the duration of an experiment can provide information on whether the experimental system and the spray device is performing as expected, and aid interpretation of data; for example nebulizer fluid may evaporate over time causing concentration of solution and a change in particle size distribution (Chen & John, 2001).

When monitoring bioaerosol particle size, concentration and biodiversity in natural environments, temporal and seasonal variations are observed (Burrows et al., 2009; Caliz et al., 2018). Replication of such natural factors in laboratory studies has been rare. However, reproducible and variable peaks in concentration of aerosol particle concentration, with consistent particle median diameter and geometric standard deviation have been achieved by varying airflow rate through a liquid bubbling aerosol generator using E. coli and Penicillium brevicompactum spores. Detailed analysis of repeatability and reproducibility was not undertaken, although limited repetitions demonstrated comparable peak concentration intensities (Simon & Duquenne, 2013).

3.4.3 Liquid bioaerosol characteristics: loading and survival

Most devices applicable for bioaerosol generation produce polydisperse size distributions. Also natural bioaerosols are generally polydisperse and understanding where the biological material predominantly resides within the particle size distribution is important for modelling efforts to understand the effects of bioaerosols. Representative experimental setups should replicate the particle size distribution generated by the natural source under investigation. Microbial viability has been found to increase with particle size (Cox, 1987; Handley & Webster, 1995; Lighthart & Shaffer, 1997), potentially due to less surface exposed to the atmosphere (Jones & Harrison, 2004), thus generating particles that are different than what is found in nature can introduce bias. Nonetheless, aggregations of cells or cells attached to non-biological particles are found in the atmosphere, where it has been shown that shielding from UV light and desiccation resulted in higher preserved viability (Clauß, 2015; DasSarmaAccepted & DasSarma, 2018). Manuscript The concentration of biological material in the aerosol divided by the concentration in the original spray fluid derives a term called the spray factor (Bowling et al., 2019). The spray factor thus measures the effect of aerosol generation process on the concentration of biological material in the aerosol. Faith et al., (2012) used spray factor to demonstrate that media composition and relative humidity influences the aerosol stability but not the infectivity of Francisella tularensis. However, spray factor can be a source of variability between laboratories as it is dependent on the sampling method. For example, sampler type, sampling fluid and distance of sampler from aerosol generator (i.e. time in aerosol phase) can affect recovery efficiency in a species-dependent manner (Marthi et al., 1991; Dabisch et al., 2012a; Dybwad et al., 2015). Care must be taken in interpretation of spray factor values between systems as the derivation is a reflection of both the experimental system (i.e. physical losses) and the susceptibility of biological material (degradation from aerosol generation, sampling and enumeration).

Understanding how differences in experimental design influence spray factor will enable selection of appropriate conditions for a particular biological material and aid inter-laboratory comparison. It would be prudent to characterize the system with a physical tracer to determine losses within the experimental system due to aerosol deposition alongside biological assessment. Examples of physical tracers include chemicals (i.e. fluorescein or uranine), bacterial spores, radiolabeled biological material or liquid, and fluorescent microspheres (Miller, 1961; Cox et al., 1970; Zhao et al., 2011; Dabisch et al., 2012b; Bowling et al., 2019). The particle size distribution formed by the physical tracer should be closely matched to the bioaerosol under examination (Bowling et al., 2019). Studies reporting spray factor should include sufficient details of the experimental system including operational parameters of both aerosol generation and sampling techniques to enable reproduction elsewhere. Variation in air and liquid flows, liquid volumes in aerosol generator and sampler, and sampling fluid composition may make inter-laboratory comparison difficult and should be reported in methodology. Comparisons between microbial species should be balanced by the knowledge that the experimental system may influence observed viability measurements (Terzieva et al., 1996; Rule et al., 2007). When it comes to culturability, diluent selection used for enumeration can affect viability (Won & Ross, 1966). It would be recommended that assessment of culturability be conducted across a range of non-selective and supplemented media and diluents whilst enumerating to ensure the microbes are not adversely influenced.

3.4.4 Dried bioaerosols: aerosol characterization from source to study

Similar issues described for liquid generated bioaerosols exist for laboratory generated bioaerosols from materials such as dried bacterial or fungal spores, pollen and dust-associated endotoxins (Thorne, 1994; Kim & Je, 2012; Afanou et al., 2014; Vimala et al., 2019). Natural bioaerosols cover the whole size range of aerosols, from a few nanometers up to 100 µm, with many of the largest sizes due to attachment to fragments of biotic or abiotic material (Löndahl, 2014; Clauß, 2015). Particle concentration and size of the composite dried particles is of utmost importance and should be characterized, preferably at the point where samples are taken for subsequent analysis. As previously mentioned, the quality and method of preparation and storage, as well as choice of aerosol generation will facilitate eas of disaggregation of clumped biomaterial and contribute to reproducibility of the aerosol (Calvert et al., 2009; Masuda, 2009). Certainly it is recommended that preparation and storage methods for dried biological material be standardized to aid flowability and reproducibility of the aerosolized material.

Few studies have been conducted comparing survival of liquid compared to dry aerosolized biological material. However, comparative studies of wet and dried generated bioaerosols demonstrate differences in aerosol survival which infer the preparation, aerosolization and rehydration during sampling of the biological material can influence downstream viability (Cox, 1970; Cox, 1971; Cox 1987; Thorne, 1994). These studies demonstrate that liquid preparations cannot be used as Acceptedfaithful representation of how dried preparationsManuscript would behave biologically.

3.5 Surrogates and biosafety

Aerosol research with pathogens requires appropriate biocontainment according to the biosafety classification of each microorganism. It is important to evaluate exposure risks of the laboratory personnel. It is recommended that aerosol chambers are kept under negative pressure, and with HEPA filter exhausts, in case of system failure. Leakage tests should preferably be performed under high pressure to assess worst case leakage rates (Perrott et al., 2017; Verreault et al., 2014). When pathogens at Biosafety Level-2 and higher are nebulized in high concentrations, additional safety precautions are required to prevent exposure (Bohannon et al., 2016; Perrott et al., 2017). Whenever possible, the use of standardized and validated nonpathogenic surrogates is recommended to facilitate aerosol studies. Examples include bacteriophages or nonpathogenic bacteria as surrogates for human or animal pathogens (Turgeon et al., 2016; Bishop & Stapleton, 2016).

Another example of surrogate requirement is representation of biodiversity of natural air samples in the laboratory for better assessment of biological detection systems or understanding occupational risk (Ratnesar-Shumate et al., 2011). The variation and concentrations of individual microbial or pollen taxa from different ecosystems is important in this context. Known concentrations of bacteria, viruses or fungal spores can be mixed and aerosolized generating a complex bioaerosol background (Ratnesar-Shumate et al., 2011; Degois et al., 2019). Berchebru et al., (2014) extended the concept demonstrating good reproducibility of production, storage and reconstitution of a standardized mixture of ten bacterial species dried under vacuum and stored for up to a year. However, replicating the biodiversity and variability of natural bioaerosols represents a major challenge and would be worth increased research to enable laboratory studies more representative of specific ecosystems.

4. Recommendations for experimental design and reporting of methodology

Understanding and rationale behind selection of an experimental system is critical for interpretation of the final data and future reproducibility. Aerosolization of biological material has a number of steps where recommendations for good research practice and increased reporting of methodologies would enhance reproducibility within bioaerosol research communities. i. Preparation and storage of material. The quality and reproducibility of the starting material influence variability of data and should therefore be standardized within a laboratory.  Species (and strain) specific effects should be considered during preparation/ storage of biological material and aerosol generation. For example, storage conditions for E. coli have been demonstrated to affect viability dependent on additives (Clement, 1961), and growth conditions (i.e. preparation method and growth phase) can influence aerosol survival (Hood, 1961; Dark & Callow, 1973; McDermid & Lever, 1996; Faith et al., 2012).  Preparation and storage methods that are most appropriate for a particular biological material must be based on an experimental rationale that should be reported in methodology.  Preparation and storage methodologies should contain sufficient detail to enable direct comparison between experiments and across laboratories.  Spray fluid composition should be characterized and reported in the methodology and matched as closely as possible to the complexity of natural phenomenology including chemical composition and concentration of biological material. ii. Aerosol generation. The complexity of natural processes makes replication of all the aspects in aerosol generation difficult. Ensuring the quality of the biological material exiting the aerosolization device and the reproducibility and accuracy of the measurement of the bioaerosol size distributions, are perhaps the most critical parts to accurately compare with the natural processes.  Design andAccepted operational parameters of the bioManuscriptaerosol generator that influence the size distribution and quality of the aerosolized material should be reported in the methodology including nozzle material and orifice size, spray pressure, spray time, volume and fluid composition.  Furthermore, for natural aerosol generation processes, understanding the impact of dynamic parameters such as the Weber number and properties such as rheology and surface tension will enable better rationale for selection of aerosol generators used during laboratory experiments designed to mimic natural outcomes.  Controls that allow physical effects of aerosol generation to be separated from biological effects should be included in experimental design. The non-biological tracer may be included in the same spray preparation as the biological material, however demonstration that the biological material is not adversely affected should be undertaken (Zhao et al., 2011) iii. Aerosol characterization. Physicochemical and biological attributes of the aerosol should be captured in the methodology to enable comparison between laboratories and support data interpretation and utilization within computational models.  Particle concentration and entire size distribution, and resulting moments such as MMAD or geometric standard deviation at equilibrium should be recorded and reported, along with the temperature and humidity. Preferably the measurements should be continuous and for the duration of the experiment.  Accuracy of sensors is critical to provision of correct measurements such as particle size, relative humidity and temperature. It is recommended that all measurement equipment is appropriately and periodically checked and calibrated against appropriate standards.

Consideration should be afforded to understanding the quality of the aerosolized biomaterial using additional techniques and assays to explore viability, injury, sub-lethal damage or death that may affect subsequent long-term characterization studies.

5. Challenges and future directions

The future prospects for bioaerosol research are bright and there will be great benefits in diverse communities interacting to solve the complex research challenges. Aerosol generation is a key component that extrapolates between the natural environment and generation of experimental data.

i. Principles and mechanisms of natural bioaerosol generation. A greater understanding of the underlying principles and mechanisms that govern natural dispersal of bioaerosol will support rational design of aerosol generators. This could result in laboratory aerosol generators that replicate the entire process or key interfaces between droplet generation mechanism, fluid composition and local environment (i.e. airflow, humidity) that epitomize the emitted natural bioaerosol (Bourouiba et al., 2014; Bourouiba, 2016; Wang & Bourouiba, 2018a; Wang & Bourouiba, 2018b; Wang et al., 2018; Poulain & Bourouiba, 2019; Poulain & Bourouiba, 2018; Poulain et al 2018; Jung et al., 2016; Gilet & Bourouiba, 2015; Traverso et al., 2013). Fluid properties and their influence on aerosol generation are less defined and should be the focus of research effort to understand their impact on both natural and experimental bioaerosol generation processes. Examples of fluid properties include rheology, surface tension, density, and associated dynamic parameters that govern the fragmentation of fluid into droplets, such as the Weber number. ii. Rational design of liquid bioaerosol generators. Empirical, theoretical and modelling tools can be used to mechanistically understand aerosol generation, aerosol output, and limit stress on organisms during generation. Understanding the interfacial physics leading to fluid fragmentation in a range of configurations, such as unsteady processes (Wang & Bourouiba, 2018b) or bubble bursting (Poulain et al., 2018), are critical to building the foundation required to quantitatively capture, and thus predict, the complex fluid processes governing spray generation. Such understanding can be integrated into high fidelity numerical fluid dynamic simulations of interfacial processesAccepted (Popinet, 2018) and to accurately Manuscript assess and optimize the environment bioaerosols experience during atomization (Fife et al., 2005; Ruzycki et al., 2013). Particular attention should be paid to areas of high velocity and complex flows that may introduce high shear forces onto biological material (Fife et al., 2005). Theoretical and computational modelling and experimentation have to be used iteratively to develop an optimized system for a given application or natural aerosolization process. A range of physical mechanisms should be considered including the interfacial physics and instabilities, shear forces applied to particle or organisms, inertial and turbulent deposition, gravitational settling and electrostatic effects. Recognizing no single aerosol generator will serve all requirements, the generation technique should be designed to meet experimental needs for reproducibility such as requisite output, droplet dispersity, generation of low background of non-biological particulates, and reproducible surface coating of spray fluid components. Ultimately the process should be as reflective of the natural process as possible. iii. Dried aerosol generation. Laboratory studies with dried bioaerosols are less prevalent than with liquid bioaerosols. Technical difficulties in preparing and storing dried biological material in ways that retain viability and support efficient aerosolization of the material represent challenges. However, parallel fields that regularly study aerosolized dry powdered material offer a rich resource of new technologies for research with dried bioaerosols. Examples include pharmaceutical drug delivery and risk assessment of occupational exposure to aerosolized nanoparticles (Calvert et al., 2009; Masuda, 2009; Tsai et al., 2012). iv. Physiological and molecular characterization of biological material during aerosol generation. A range of aerosol generation techniques exist (Table 1) and understanding the effect of each aerosol generation mechanism on the particular biological material investigated would enable rational selection of an appropriate device that minimizes damage. Damage could occur at the sub-lethal level and therefore, alongside traditional culture-based methods, orthogonal techniques for investigating bioaerosols should be used. Microbiology and virology analysis methods offer a variety of assays that target specific cellular functions by dye inclusion/exclusion or PCR methods for genetic analyses (Stone & Johnson, 2002; Zhen et al., 2013; Zhen et al., 2014; Alsved et al., 2018; Turgeon et al., 2014; Allegra et al., 2016). Molecular tools are progressing rapidly and are being implemented to interrogate bioaerosol diversity and complexity (Brodie et al., 2007), as well as survival and activity during aerosol generation and transport (Ng et al., 2018; Šantl-Temkiv et al., 2018). Molecular techniques could be useful investigational tools for deconvolution of the relative importance of interacting stresses during aerosol generation, transport and sampling that occur within experimental procedures and that may differ between natural processes. Furthermore, researchers should continually evaluate advances in assay development for probing prokaryotic and eukaryotic physiology (Cao-Hoang et al., 2008) to better understand the impact of aerosol generation and facilitate selection of the most appropriate aerosol generation mechanism for a particular bioaerosol. v. Polydispersity of aerosols. Natural and experimental bioaerosols are generally polydisperse; however this can present problems in understanding particle size dependent phenomena. Monodisperse generators such as the droplet-on-demand technologies enable refined exploration of aerosols by reducing variability within individual droplets. Next generation dispensers that extend the range of applications for bioaerosols would be beneficial with properties such as operability with highly viscous, complex biological fluids and capacity to generate smaller droplet sizes. vi. Surrogate biomaterial and spray fluid. Surrogates can be used to understand characteristics of hazardous aerosolized material that is needed to develop risk management strategies (Sinclair et al., 2012; Berchebru et al., 2018; Turgeon et al., 2014; Bishop & Stapleton, 2016). Selection of an appropriate surrogate should be tailored to the properties investigated such as aerodynamic properties (Phillpotts et al., 2010; Turgeon et al., 2016; Dybwad et al., 2017). However, care must be takenAccepted in extrapolating data between surrogate Manuscript and infectious material to prevent under- or over-estimation of risk (Sinclair et al., 2012). Ideally, parallel studies should be performed that evidence extrapolation between the surrogate and the pathogenic material. Numerous studies have developed respiratory secretion liquids that simulate the chemical composition of natural secretions (Bose et al., 2016; Pytko-Polonczyk et al., 2017), and these may be used as spray fluid surrogates in aerosol studies. However, research is required to better understand and reflect on effects such as the health status and subject variability which can influence the respiratory secretion composition (i.e. mucin type and content). Furthermore, research to better identify and standardize surrogates would benefit communities involved in risk management. Indeed, studies using human or cell line derived respiratory secretions have demonstrated increased survival of influenza virus deposited as droplets onto banknotes (Thomas et al., 2008a) or as aerosol droplets (Kormuth et al., 2018; Kormuth et al., 2019). vii. Understanding and representing variation and diversity in bioaerosols. Natural bioaerosols are complex mixtures of many microbial species in addition to other biological components (i.e. allergens, endotoxins, and glucans) and abiotic material (i.e. dust). Understanding the health effects of airborne bioaerosols such as allergens would be supported by correlative laboratory studies (Douwes et al., 2013). Microbiome analysis has indicated the large biodiversity present in aerosols (Caliz et al., 2018; Li et al., 2019; Mescioglu et al., 2019), however, few studies have attempted to replicate this complexity in the laboratory (Ratnesar-Shumate et al., 2011; Degois et al., 2019). Including representative dried abiotic material, for example, dust from livestock or composting facilities, is advantageous in generating a more realistic bioaerosol. Some studies have replicated this complexity by collecting samples from the natural environment including the microbiological flora to use in laboratory aerosol studies. For example, compostable waste, water from lakes and seas, and soils for replicating splashes from rain drops have been aerosolized in laboratory settings (Heldal et al., 2001; Aller et al., 2005; Joung & Buie, 2015; May et al., 2017; Joung et al., 2017). viii. Droplet microfluidic platforms. Microfluidics though underutilized within the aerosol community, offer an attractive platform for the development of novel droplet generators and understanding the complexity of natural droplet formation (Cole et al., 2017; Metcalf et al., 2018). Favorably, monodispersity can be achieved with microfluidic nebulizers due to the confined geometry of the microfluidic channels (Anna, 2016). ix. Diversity of aerosol research. A major challenge is the diversity of aerosol research and the translation of advances in related areas that could benefit bioaerosol research. Opportunities created through conferences, symposia and meetings that bring together different communities will facilitate awareness, interaction and integration between fields. Pertinent examples include advances in physics of complex fluid fragmentation, pharmaceutical delivery methods, microfluidics, and molecular analysis techniques. Structured training of aerosol scientists to incorporate interfaces to other disciplines would facilitate multidisciplinary knowledge and skills.

6. Conclusions

Bioaerosol research is challenging due to the number of points within the experimental system where variability can be introduced and due to the dynamic nature of aerosol formation and evolution over time and distance. Despite this, recommendations are proposed for best research practice and rigor in methodological reporting of bioaerosol research. This aims to minimize variation in bioaerosol data, aid data interpretation by experimenter/ reviewer, and prompt greater representation of the natural aerosol generation process that is researched. Examples include consideration and characterization of the starting material, a robust rationale for selection of an aerosol generator, consideration of the impact of the aerosol generation mechanism has on the biological material and appropriate characterization and reporting of the output aerosol.

Developments in related research fields will undoubtedly offer a rich scope for designing aerosol generators that are specific to a research challenge, and to improve our understanding of the impacts naturalAccepted and experimental generation techniques Manuscript have on biological material. Efforts should be placed on maintaining and enhancing the multidisciplinary skills required in future bioaerosol research.

Acknowledgements

The authors acknowledge organizers of this special issue “Bioaerosol Research: Methods, Challenges and Perspectives”, including Shanna Ratnesar-Shumate and Alex Huffman, based on requests from the Bioaerosol Working Group and after discussion during the Bioaerosol Standardization Workshop at the International Aerosol Conference in St Louis, Missouri in September 2018. RJT and STP acknowledge the Ministry of Defence, UK for provision of funding supporting generation of the review. LB acknowledges support by the MIT-Lincoln Laboratory, Ferry Fund, and the Richard and Susan Smith Family Foundation. MA and JL acknowledge the Swedish Research Council FORMES and AFA insurance for funding.

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Accepted Manuscript Figure 1. Natural and experimental mechanisms of bioaerosol generation.

Accepted Manuscript

Table 1. Common methods for generation of experimental bioaerosols.

Aerosolization Example Properties of Experimental Particle Airflo Fluid Material References mechanism1 device(s) material considerations size2 w flow recirculation (L/min (mL/mi ) n) Liquid atomization Twin-fluid Collison Liquid Fluid forces and 0.032 – 1.3 2 – 7 0.02 – Yes May, 1973; Liu & Lee, 1975; atomization nebulizer suspension recirculation µm (GSD (per 0.08 Reponen et al.,1997; Stone & (pneumatic may damage 1.8)3 jet) Johnson, 2002; Wang et al., recirculation) some biological 2004; Yao & Mainelis, 2006; material Grinshpun et al., 2007; Rule et al., 2009; Turgeon et al., 2014; Zhen et al., 2014; Ibrahim et al., 2015; Bowling et al., 2019 Twin-fluid Sparging Liquid Fluid forces may <5 µm 2 - 30 2 Generally Reponen et al., 1997; Mainelis atomization liquid aerosol suspension damage some no4 et al., 2005; Rule et al., 2009; (bubble bursting) generators biological Simon et al., 2011; Simon et al., (SLAG) material 2013; Zhen et al.,2014 Twin-fluid Centered Liquid Fluid forces may <2 µm 0.3- 0.2 Generally Zhen et al., 2014 atomization flow; Single suspension damage some 1.5 no4 pass biological aerosolizer material Twin-fluid Commercial Liquid May recirculate <3.5 µm <8 8 Yes/ No Najlah et al., 2014; Astudillo et atomization/ Jet nebulizersAccepted suspension material; Manuscript venturi al., 2018; Pritchard et al., 2018 nebulization (e.g. principle Sidestream nebulizer, Pari LC Sprint) Twin-fluid Flow focusing Liquid Favourable 9 – 100 µm 300 – 0.035 – No Ganan-Calvo & Gordillo, 2001; atomization (flow- monodisperse suspension monodispersity; (initial wet 20005 0.5 Martin-Banderas et al., 2005; focussing) aerosol particle size distribution Thomas et al., 2008b; Thomas generator depends on ) et al., 2009; Duan et al., 2016 (FMAG) pressure and flow rate Centrifugal Spinning top Liquid Generate larger 0.95 - 6.7 N/A 1 No May, 1949; May, 1966; Ellison, nebulization aerosol suspension particle sizes; µm 1967; Young et al., 1974; generators size range is Mitchell, 1984; Eisner & (STAG) controlled by Martonen, 1988; Melton et al., modifications 1989; Biddiscombe et al., 2006; such as Bohannen et al., 2015 impaction of material prior to exit from device Ultrasonic Commercial Liquid Conversion of 1 - 15 µm <100 0.001 – No Berglund & Lui, 1973; nebulization6 nebulizers suspension high frequency (initial wet where 2.2 Wiedmann & Ravichandran, (acoustic waves; (e.g. sound waves droplet size applic 2001; Katial et al., 2012; Najlah vibrating mesh or SonotekTM, into mechanical 10 – 60 able et al., 2014; Ratnesar-Shumate orifice generation) DeVilbiss energy that µm) et al., 2011; Santarpia et al., Pulmosonic, disrupt liquids 2012; Dybwad & Skogen, Omron into droplets. 2017; Dabisch et al., 2017 microair, Rhône- Poulenc Fisoneb) Vibrating mesh Commercial Liquid May be active or < 6 µm <4 N/A No Turgeon et al., 2014; Najlah et nebulization nebulizers suspension passive. Piezo- al., 2014; Allegra et al., 2016; (active or passive) (e.g. Omron, element vibrates Gowda et al., 2017; Pritchard et Pari, a mesh substrate al., 2018; Astudillo et al., 2018; AeronebAcceptedTM) contacted Manuscript by Bowling et al., 2019 liquid. Movement pushes liquid through mesh into airstream. Electrospray Model 3480, Liquid Very small 0.002 - 2 <2 0.008 – No Mulholland et al., 2001; Kim et ionization TSI Inc suspension droplets µm (initial 0.03 al., 2008; Eninger et al., 2009; generated; wet droplet Guha et al., 2012; Almeria & Reduced virus size 0.13 – Gomez, 2014; Rutkowski et al., and protein 5 µm) 2018; Ganan-Calvo et al., 2018 agglomeration Pulsed droplet Ink jet Liquid Very 10 – 400 N/A 0.0001 – No Ulmke et al., 2001; Amirzadeh ejection droplet-on- suspension reproducible µm (initial 0.005 & Chandra, 2010; Minov et al., (piezoelectric or demand monodispersity; wet droplet 2014; Harris et al., 2015; Vaugn pneumatic) dispensing Large initial size) et al., 2016; Ionkin & Harris, technology droplets 2018; Fernandez et al., 2019 controlled by orifice size Solid powder Air flow dispersal Powder Powdered/ Airflow on Depends on <5 - N/A No Cox et al., 1970; Reponen et al., (i.e. ejector or disperser/ dried material powder/ surface material >15 1996; Reponen et al.,1997; Lee venture) Agar tube or fungal size and et al., 2008; Tang et al., 2008; disperser spores on quality Jung et al., 2009; Tsai et al., agar surface 2012; Tiwari et al., 2013; Pokharel et al., 2019 Air flow dispersal Swirling flow Dried Stable aerosol As above 15 N/A No Reponen et al., 1997; Afanou et disperser materials or concentrations al., 2014 fungal spores of smaller spores on agar surface Scraping/ mixer Rotating Powdered/ Packing density As above <15 N/A No Calvert et al., 2009; Masuda, brush dried material can vary output 2009; Verreault et al., 2012; generator scraped by Tsai et al., 2012; Wang et al., (RBG) blade or 2014 Acceptedimpeller Manuscript Fluidized bed Vilnius Powdered/ Range of aerosol 0.5 – 40 <10 N/A No Marple et al., 1978; Prenni et aerosol dried material concentration µm al., 2000; Calvert et al., 2009; generator forced and particle Masuda, 2009; Tsai et al., 2012; (VAG); PITT- through bed sizes Harnish et al., 2014 3 of heavy particles fluidized by air Note: Values in table are for standard settings of aerosol generator. For many generators there is a possibility to vary output by changing operational parameters including flow rate (gas or liquid), disc rotation speed, orifice size and vibrational frequency. 1 Details of mechanisms can be found within references. 2Droplet sizes are derived from references. It is often not denoted whether these represent initial or equilibrium sizes. It is anticipated they represent equilibrium particle size unless stated. 3Larger dried particles such as fungal spores and beads can be generated up to approximately 5 µm. 4Some generators operate in both recirculating and non-recirculating modes. 5Pressure in millibar. N/A, not applicable. 6Vibrating orifice aerosol generator (VOAG) is no longer commercially available; however the mechanism is well documented as a means of generating precise aerosol concentration standards in laboratory studies.

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