I hope you're doing well and enjoying the end of summer. I also hope that you'll find the following article about fungal aerosols by Dr. Harriet Burge both interesting and useful.

With best wishes,
Dave Gallup

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Introduction
The development of a strategy or plan for documenting fungal growth in a building depends on a knowledge of how, where, and why fungi grow in buildings. With this knowledge one can usually find fungal growth and possibly go directly to recommendations for its removal. However, in some cases it is necessary to collect aerosol samples. Reasons for collecting samples of fungal aerosols include a need to actually document exposure, to assess whether or not identified fungal growth is producing aerosols, to check the possibility that hidden growth has occurred, or to satisfy a client (or his/her attorney) that you have done a complete investigation.

If you are going to understand the data recovered from air samples, it is essential that you have an understanding of the nature of fungal aerosols, their sources and mechanisms for aerosolization.

The nature of fungal aerosols
Aerosols are suspensions of solid particles or liquid droplets in air (or other gas) that remain airborne for at least minutes. Fungal aerosols are parts of fungal bodies that are small enough to become airborne. They are usually spores, but other parts of the fungal body may be a part of fungal aerosols (Reponen, et al., 2007). Fungal spores, especially those of ascomycetes and basidiomycetes, comprise a large proportion of the outdoor coarse particle aerosol (1-10µm). In tropical rainforests, these fungi make up 35% of this aerosol on average, and estimates for extra-tropical areas are similar (Elbert, et al., 2007). Although studies have not been done for dry spores such as Cladosporium, percentages in this particle size range are likely to be similar given the recorded concentrations of these fungi.

How long the particles remain airborne depends on their density, their aerodynamic diameters, air movement, and the nature of the immediate environment.

We usually assume that spores have "unit density," which means that they have the same density as water. This sounds reasonable since water fills the interior of the spore. However, this assumption introduces a variable error in estimations of removal rates (Reponen, et al., 2001).

Aerodynamic diameter is the diameter of a smooth sphere of unit density that would fall in still air at the same rate as the particle (spore) in question. If you have a perfectly spherical spore with no surface ornamentation, then its actual diameter is usually close to its aerodynamic diameter. Few spores are either completely spherical or smooth. Thus, generally, aerodynamic diameter must be determined experimentally. One way this is done is to use cascade impactors that are designed to collect particles in specific ranges of aerodynamic diameters. Data from several sources using this method indicate that the common fungi involved in indoor growth have aerodynamic diameters in the range of 1.5-7µm. Many common Aspergillus species fall into the lower part of this range (Meklin, et al 2002). Practically, we often use the smallest diameter of a spore as its aerodynamic diameter. This assumes that the spore becomes aligned with its long axis parallel to forces affecting its movement (McCartney, et al., 1993). We often ignore surface ornamentation, assuming that this introduces only small errors. However, empirical data to test this hypothesis is not available.

Apparently most fungal spores do not increase in volume with increasing relative humidity until the humidity reaches near 100%. Then, the increase is relatively small (Reponen, et al., 1996). However, Madelin and Johnson (1992) report an increase in aerodynamic diameter when spores are passed through air at 98% RH and 38°C, compared to 40% RH and 20°C. This difference points out the importance of considering comparability of methods used when evaluating different studies, as well as the kinds of spores.

In addition to spores, fungal aerosols include fragments of spores and mycelia (Reponen, et al., 2007). However, tests documenting the concentrations of these particles have involved aerosolization from either cultures or building materials using air speeds much higher than would be likely in an indoor environment (20 l/m) and sample collection modalities that also produced high flow rates that could have caused fragmentation. It is also not clear that spores are being fragmented rather than mycelium. Whether this makes a difference with respect to exposure to fungal agents of human diseases (ie. allergens or toxins) is unknown. Obviously, fungal fragments smaller than cell size are not important with respect to infectious disease (Seo, et al., 2007 & 2009).

Sources for fungal aerosols
Outdoor fungal aerosols are derived primarily from fungal growth on plant surfaces (the "phylloplane" fungi) or from fungi growing on the surface of the soil (e.g. mushrooms, puffballs, and cup fungi). Fungi that grow on leaf and stem surfaces are often pathogenic for the host plants. The kinds of fungi in the air depend on the kinds of plants that are common and nearby. Intensive (or extensive) agriculture may provide vast acres of plants susceptible to a particular fungus, which will then produce spores that may dominate the outdoor fungal aerosol, especially for those crops that are harvested after the leaves have died (Mitikakis, et al., 2001). Likewise, fungi that grow on the ground are often dependent on symbiotic relationships with particular plants and will be abundant in areas where those plants grow.

Fungi that grow within the soil (the so-called "soil fungi") may become airborne during windstorms or when the soil is actively disturbed, such as during construction activities. Coccidioides immitis is a human pathogen that becomes airborne from desert dust during windstorms, causing epidemics of human disease. The composition of soil fungal communities is related in part to precipitation. In dry weather it appears that fungal communities become diverse and stable, while abundant rainfall will tend to destabilize the communities, and a few fungi will become dominant. This fact has implications for changes in carbon cycling in the soil as a result of climate change (Hawkes, et al., 2011).

Composting processes utilize soil fungi and bacteria to decay organic material that is often gathered into concentrated piles over many acres of ground. Measurements of fungi released from actively disturbed compost (as occurs during turning) range from 10⁴ to 10⁵ spores/m³ (Taha, et al., 2007).

Sources for fungal aerosols indoors include outdoor air (the predominant source) and growth on indoor materials. If indoor spaces received as much water as outdoors, then our buildings would be filled with fungi. We all know that just a relatively small leak can initiate fungal growth and flooding, such as happened in New Orleans and is occurring now along the Mississippi River, provides sufficient water to stimulate growth on all interior surfaces. Although we think of indoor fungi being different than those outdoors, they really are not. All indoor fungi came originally from outdoor sources. The reason some fungi do especially well indoors is that they find food under conditions of relatively little competition.

The generation of fungal aerosols
The first step in aerosol generation is the formation and release of fungal spores. For many (perhaps most) spores, internal clocks have evolved that control the timing of these processes. This insures that spores will be produced and released at times that are most likely to provide appropriate conditions for spore transport to a suitable substrate for growth (Bell-Pedersen, et al., 1996). Pathways to spore production are beyond the scope of this article. However, methods by which spores are released from spore bearing structures are an integral part of aerosol formation.

External mechanisms for spore release
External mechanisms for spore release involve the nature of the physical attachment of the spore to the fungal body, and bonding forces that tend to keep spores attached to the surface where the fungus is growing. Sharp drops in relative humidity appear to play a large role in release of spores that do not have internal spore release mechanisms (Jones & Harrison 2004). Wind also plays a role in breaking bonds between spores and spore-bearing structures as well as those that bind spores to leaf surfaces after release.

Additional forces that can cause "passive" spore release are rain splash, mechanical activity (such as occurs during harvesting of field crops), or by remediation activities. Splash dispersal involves the action of raindrops hitting a reservoir of fungal spores and actually splashing them into the air. Cladosporium carpophilum, a pathogenic species of peach and other fruits, is splash dispersed from infected twigs (Lan & Scherm 2003). Spores of puffballs (basidiomycetes) are also splash dispersed. Indoors, splash dispersal might occur when water sprays are directed toward moldy surfaces, or within humidifiers where fungi are growing on surfaces at the air/water interface.

Indoors, it appears that air speed is generally too low to release most spores from their growth sites. The infamous fungus, Stachybotrys chartarum, has been studied for air speeds that will release spores from conidiophores on surfaces (Tucker et al., 2007). Micronewton forces were needed to dislodge spores from undisturbed colonies. Using a microflow apparatus, they determined that most spores that are released at a certain airflow enter the aerosol within 5 minutes, and that following this time 99% of the spores remain attached. They point out that airflows in indoor environments are generally much lower (in the nanonewton range) and air movement is unlikely to be effective in removing Stachybotrys spores from a colonized surface.

Penicillium and Aspergillus spores may be released at relatively low air velocities (0.4 m/s). However, Cladosporium spores are not released at this speed. Kildeso et al. (2003), studied release of spores from wet building materials. They determined that such release is dependent on many factors including humidity, nutrients, interaction between microorganisms, and possible peak airflows. They conclude that because all of these factors are involved, area covered by fungal growth does not predict potential exposure. Kanaani et al. (2009), provide a good review of the laboratory studies evaluating relationships between environmental conditions and release of fungal spores from culture.

Intrinsic spore release mechanisms
Intrinsic spore release mechanisms are powerful, and can shoot small (micron sized) spores distances of 1000 times the spore diameter (Schmale et al., 2005). Water relationships play an important part in most intrinsic spore release mechanisms.

Basidiospores
Basidiospores have evolved a unique bilaterally symmetrical shape that facilitates an especially interesting method of spore discharge mechanism. This mechanism can shoot the spores as far as 2mm, which is far enough to place the spore in the space between gills so that it can fall into the moving air stream (Fischer et al., 2010). The mechanism is powered by "Buller's drop" which forms at the base of the asymmetric spore and moves rapidly across the spore surface leading to a catapult motion that discharges the spore (Stolze-Rybczynski et al., 2009). Noblin et al. (2009), performed elegant experiments that elucidate the stages in this process.

Ascospores
Ascomycetes, on the other hand, often use a rocket mechanism (in essence, pressurized squirt guns) that shoot spores as far as 2 or more meters (Yafetto et al., 2009). The limiting factor for the distance that spores can travel after forcible discharge is drag, which is essentially the resistance caused by rapid travel through air. The larger the spore, and the more aerodynamic the shape, the further the spore can travel (Yafetto et al., 2008). Ascospore shapes have evolved to minimize drag. Roper et al. (2008), have experimentally determined that spore shapes in ascomycetes have evolved so that drag does not exceed 1% of the minimum possible. Thus many ascospores are oval or threadlike. The actual mechanism that triggers the squirt gun process of many ascomycetes varies. Osmosis certainly plays a role by rapidly filling the ascospore sacs with water, which ruptures the apex, causing the spores to shoot out. Some ascospores are released in groups, which increases the mass of the projectile, enabling it to travel further. On the other hand, the ascomycete Venturia inaequalis, a plant pathogen, the spores of which are often abundant in outdoor air, releases spores in response to raindrop-induced vibrations of the leaves on which the fruiting bodies are growing (Alt & Kollar 2010). Sclerotinia sclerotiorum synchronously discharges ascospores in a way that sufficient airflow is produced to propel the spores through the boundary layer of still air that surrounds the fruiting body and into the moving air stream (Roper et al., 2010).

Conidia
The asexual spores also have intrinsic mechanisms for spore release. Electrostatics plays an important role in discharge of many fungi, including Stemphylium botryosum, Pyricularia oryzae, and Drechslera turcica (Leach 1980). Leach documented this in part by neutralizing spore discharge using positive ions. Adams et al. (1986), documented that asexual powdery mildew spores were released with rapid drops in relative humidity, indicating some kind of intrinsic mechanism. The explosive formation of gas bubbles may launch spores that then travel millimeters away from the parent fruiting body (Meredith 1963; Fischer et al., 2010).

Aerosol transport and concentrations
Factors affecting outdoor transport and concentration changes
Maximum aerosol concentrations will be seen close to sources. These concentrations are dependent on diurnal patterns of spore production and release, as well as meteorological and other factors that force spores into the air. The transport of fungal spores depends first on the escape of the released spore from its immediate surroundings. Studies of soybean pathogens indicate that the spore escape rate depends on turbulence in and above the canopy of plants surrounding the release site, and the filtration accomplished by impaction on surfaces within the canopy (Andrade et al., 2009).

Once outside the canopy, spore concentrations depend on transport mechanisms (wind) and deposition mechanisms (gravity, drag, rain, and impaction on surfaces). Gravity has only a relatively small impact compared to other factors on most spores outdoors. Aerodynamic drag rapidly slows small spores in still air, but in windy conditions the spores can be transported long distances. Rain essentially washes dry spores from the air. Wet spores are probably also removed, but the strong activation of sources during rain may mask this effect.

Fungal spores clearly can be transported long distance and can survive for many (even hundreds) of years. Charles Darwin collected dust from over the Atlantic Ocean during his travels during the 19th century, and that dust, analyzed in the 21st century produced culturable fungi and bacteria that had been transported from distant land masses (Gorbushina et al., 2007).

Distances traveled can be very small or very large. Spore transport is likely to follow prevailing winds. For the plant pathogen Leptosphaeria maculans, spore deposition as measured by infection rates declined 50% within 12 meters. This measurement was, of course, affected by viability. In other words, if spores die quickly, their transport will not be measured by this method (Guo & Fernando 2005). On the other hand, the relatively large sporangia of Phytophthora infestans (potato blight) were collected using spore trapping methods 500 meters downwind of an infected site (Aylor et al., 2011).

Clearly, meteorological variables strongly affect spore transport as well as spore release as discussed above. This topic has been extensively reviewed by Jones and Harrison (2004). Their paper is strongly recommended to those who are especially interested in the topic of fungal aerosols.

Indoor/outdoor relationships
Indoor/outdoor relationships have long been of interest to those studying indoor mold concentrations. In fact, one of the most common ways for estimating the probability of indoor growth has been the use of indoor/outdoor ratios in one form or another. However, studies that actually document how spores move from outdoors to indoors are not common. It is generally true that indoor and outdoor spore concentrations and populations are correlated with the indoor concentrations usually lower than those outdoors. Indoor concentrations that exceed those outdoors may result from indoor growth, but also from past penetrations of concentrated outdoor aerosols. On the other hand, indoor concentrations that are lower than those outdoors may, in fact, indicate a "clean" indoor environment, but may also be related to slow penetration of extant outdoor aerosols. Li & Kendrick (1996) used path analysis to study the relationships between different kinds of fungal spores indoors and out. They determined that, overall, indoor and outdoor concentrations were strongly correlated, although Aspergillus and Penicillium did not appear to be correlated. Indoor/outdoor relationships differ between May-October and November-April.

Spore penetration into a full-scale model chamber was found to depend on pressure differences, rather than air leakage unless a clear path was present (e.g., open windows or doors) (Airaksinen et al., 2004). Those authors point out the importance of these facts with respect to ventilation design.

Aerosol transport and concentration dynamics indoors
The transport of fungal aerosols within a building and changes in concentration over time are dependent on the source of the aerosols, aerodynamic spore sizes, patterns of airflow within the building, and opportunities for deposition. Aerosols from surface growth within a room are likely to be most concentrated within that room, and may be completely absent from other rooms that are at a positive pressure with respect to the moldy room. Even if the moldy room is positive with respect to the rest of the building, the concentrations will still tend to decrease as distance from the source decreases. With adequate ventilation of a moldy room, spore concentrations will gradually decrease as the source strength diminishes over time.

Few studies have actually documented transport of fungal aerosols through buildings. Slightly moldy studs did not release a significant number of spores under a variety of conditions designed to encourage their release (Rao et al., 2009). Kanaani et al. (2008), studied deposition (removal) rates for fungal spores in a room-sized chamber and found that spore size was important as well as ventilation. It is interesting to think about these facts in light of the more common recovery of Penicillium and Aspergillus spores, than those of larger-spored fungi (e.g., Alternaria, Epicoccum, etc.) known to grow on indoor materials. This is one of the biases imposed on the use of air sampling to determine the status of a building with respect to fungal growth.

Conclusions
The information presented here represents merely the tip of the iceberg of the information available on fungal spore aerosols. Most of the available literature has resulted from plant pathology research, although in most cases, the principles and the models developed are probably widely applicable. Clearly, the indoor environment has been poorly studied with respect to factors that lead to the formation and transport of fungal aerosols. This should be a fruitful field of research for new investigators.

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