Passive Release of Fungal Spores from Synthetic Solid Waste Surfaces

Passive release of fungal spores can occur from various natural and anthropogenic sources leading to significant concentrations in ambient air with potential effect on health and climate. The estimation of fungal spore release is a critical parameter necessary for the realistic assessment of health risk using dispersion models and in global climate modeling. This paper presents results from experiments conducted to seek a better understanding of the process of passive fungal spore due to wind. Laboratory studies were conducted to measure emission fluxes of a test fungal species (Penicillium chrysogenum) grown on two test surfaces (aluminum foil and cardboard) in a flux chamber in response to air flow. Spore growth on the aluminum foil correlated with the amount of nutrient, while for cardboard, fungal growth was observed just with the presence of water without any external nutrients. The released spores were collected using a commercially available impinger (Biosampler) and quantified using fluorescence microscopy. Spore flux correlated positively with the number of spores on the surface and with air velocity above a threshold velocity. Fungal spore flux decreased on continuous exposure to air. Microscopic inspection of the surface revealed that the normally upright hyphae bearing the fungal spores collapsed after exposure to air thus suggesting that the decrease in flux was due to a decrease in the aerodynamic drag on the spores. Fungal hyphae also collapsed when there was depletion of nutrients for spore growth leading to reduction in flux. A preliminary mathematical model that estimates spore flux based on the energy transfer between the air and the fungal spores and the energy required to remove the spores is presented. A characteristic energy parameter for aerosolisation was obtained from the model fit. Using this parameter, the model predicted experimental fluxes under various conditions reasonably well.

Bioaerosols in the atmosphere pose potential health hazards such as allergy, asthma, bronchitis, dermatitis and diarrhea (Lavoie et al., 2006;Taha et al., 2006;Park et al., 2012).Deleterious health effects are possible even when the bioaerosols are non-viable (Griffiths and DeCosemo, 1994).The role of microorganisms in influencing climate has also received recent attention.The influence on ice/cloud condensation nuclei has been reported in studies conducted in laboratory as well and higher altitude measurements (Burrows et al., 2009;Sesartic et al., 2012).From the perspective of environmental risk assessment or climate modeling, emission rate or flux (source term) is a critical input parameter in bioaerosol dispersion models or climate models.Therefore the estimation of the flux or rate of bioaerosol release or emissions from different sources is essential.Emission fluxes can be estimated from available experimental measurements or obtained using a reliable process model.
Release of bioaerosols, especially fungal spores, to air from a source occurs via two processes -active and passive release (Górny et al., 2001).The process of active release is triggered by an internal mechanism that is specific to the biological species and certain external environmental factors such as relative humidity or temperature.Passive release is induced by external mechanical forces such as wind and can potentially occur continuously under different environmental conditions.There have been a few field measurements of bioaerosol fluxes from natural surfaces obtained using the gradient measurements (micrometeorological method) of aerosol concentration (Burrows et al., 2009).These measurements however do not distinguish between passive or active release.In order to develop a reliable process model to characterize passive release of bioaerosols, it is necessary to understand the process under controlled conditions.Passive release of various bioaerosols have been studied previously using a variety of external mechanical forces such as physical vibration (Leach et al., 1982), rain drops (McCartney, 1991) and sudden gusts of wind (Legg, 1983).Bioaerosol release from composting activities under control and field environments using a pilot scale wind tunnel was also reported (Taha et al., 2005b(Taha et al., , 2006)).
Fungal spores constitute a significant portion of bioaerosols measured in ambient air and are released both passively and actively from a large variety of fungi.The present study focuses on the passive release of fungal spores with wind as the external agent.When fungi grow on a surface, a mat-like structure known as mycelium is usually formed on the surface.When conditions are unfavorable for vegetative growth, vertical structures called aerial hyphae (or conidiophores) are formed with spores (conidiospores) at their ends.There are several reports of passive release in indoor environments from surfaces such as gypsum boards in buildings, ceiling tiles or air conditioning ducts (Górny et al., 2001;Kildesø et al., 2003, Sivasubramani et al., 2004), examining the influence of velocity, agitation, and relative humidity on the passive release of bio-aerosols from different surfaces under controlled laboratory conditions.Kildesø et al. (2003) used a rotating air source to aerosolize and measure fungal spores grown on a gypsum board using an aerodynamic particle sizer (APS) and observed variation of fungal spore flux with fungus type.Madsen et al. (2012) using a similar setup observed that spore release increased with decreasing air relative humidity.Gorny et al. (2003) used a specially designed flow through chamber to measure fungal spores release from an agar surface and a ceiling tile using an optical particle counter.They observed that the release was dependent of the morphology of the fungal colonies grown and on surface features such as indentation and roughness.A threshold velocity was also required to cause spore release.Sivasubramani et al. (2004) used a fungal spore source strength tester (FSSST) to induce aerosolisation of fungal species grown on different indoor surfaces and measured the spore release using an impinger.Variation of fungal spore fluxes as a function of surface type was observed and was attributed to surface dependent spore densities and binding strengths.Another notable observation was the decrease in spore flux after the first few minutes of relatively high release.This was attributed to the relatively easier release of all mature dry spores initially, whose binding on the surface was assumed to be weaker than that of immature spores.Timmer et al. (1998) examined the release of fungal spores on leaves in an environmental chamber observed that release was enhanced when relative humidity is suddenly increased or if there was rain or other vibration.Su et al. (2000) reported that a drying environment resulted in an increase of spore release.
From literature on passive release of fungal spores, it is clearly established that air velocity has a direct positive correlation with spore release beyond a threshold velocity.There are several issues related to spore release that require further investigation.One of these is the decrease in spore flux decrease with prolonged exposure to air with a tentative hypothesis of spore maturity levels.There have been contrasting reports on the effect of relative humidity on spore flux that needs resolution.From the literature reports it is unclear whether the change in relative humidity influences the spores physiologically causing changes in release mechanism or if it is the drying and wetting that causes the associated changes in spore binding to the hyphae.The effect of the surface type on spore release was also observed.This was attributed to the binding of the spores to the surface.In the case of fungi, the spores are not directly attached to the surface.This implies that the role of the surface may not be as a result of surface binding.These issues need to be resolved for better understanding of the mechanism of fungal spore release, especially for the development of a robust physical mathematical model.Geagea et al. (1997) reported a mathematical modeling effort in this direction by estimating drag forces on spores due to air flow.
In the present study, the objective is to experimentally investigate some of the unresolved issues related to passive fungal spore release.Laboratory experiments are conducted to measure fungal spore release in a laboratory scale flux chamber and to investigate the effects of air velocity, surface type, incubation time (or age) and surface spore density on the spore release.The modeling approach of Gaegea et al. (1997) is also extended to develop a simple mathematical model with one fit parameter to estimate spore flux from surfaces.The objectives are directed at providing a better understanding of the mechanism of passive release under the influence of wind and provide a possible mathematical representation of fungal spore release in various scenario.Though the experiments and the analysis are applicable for the passive release of fungal spores in general, specific elements of the experimental protocol were selected with reference to the release of fungal spores from municipal solid waste, which contain nutrients to sustain fungal growth.

Measurement of Bioaerosol Flux
Flux experiments were conducted using an experimental setup shown schematically in Fig. 1.The flux chamber consisted of top and bottom sections separated by a sealant layer that facilitated the creation of an air gap of desired thickness.The bottom section contained the test surface on which the funguswas grown.For all the experiments described here, the height and the width of the air gap was maintained at 3 mm and 50 mm respectively.Aerosolisation of the spores was achieved by providing a flow of clean air from a compressor through a HEPA filter at a desired flow rate.The aerosolized spores from the test surface were sampled using a specialized impinger -Biosampler ® (SKC Inc.) containing a phosphate buffer solution with 0.01% Tween80 (PBST).The volumetric airflow rates varied between 18 L min -1 and 117 L min -1 , yielding linear air velocities in the range of 2 m s -1 to about 13 m s -1 above the test surface.These velocities were in the range of observed ambient air velocities that open municipal solid wastes are exposed to.Since, the Biosampler ® was designed to sample at a fixed flow rate of 12.5 L min -1 , the excess flow was vented through a fumehood via a disinfectant solution.The emission flux, N, was estimated using Eq. ( 1), where A is the area of test surface and ∆t is the time interval of spore collection, which was typically 30 minutes.The spores collected in the Biosampler ® during a given sampling interval were measured by processing all the liquid in the impinger.The total number of spores aerosolised from the surface was obtained by multiplying the count estimated from the impinger sample (using microscopy) by a scaling factor equal to the ratio: flow rate through the flux chamber/flow rate through the impinger.Additionally, at the end of each experimental run, the top plate of the flux chamber and the tubes connecting to the Biosampler ® were rinsed to collect all fungal spores that might have deposited on the walls of these surfaces and counted.The final corrected value of the aerosolized spores was obtained by adding the scaled spore density collected in the impinger and the spores deposited in the experimental system.This value was used in equation 1 to obtain the bioaerosol flux.

Preparation of Test Surface
The fungal species selected for this study was Penicillium chrysogenum (PC), which is a representative saprobe of the genus Penicillium that is a dominant species present in ambient air, especially associated with organic solid waste (Taha, 2005a).This particular benign species was therefore chosen as a suitable safe surrogate for conducting laboratory studies in a controlled environment.Specimens of PC were obtained from microbial type culture collection (MTCC, India, catalog No.6795) and were cultured on Czapek yeast extract agar (CYEA) with a composition of sucrose: 30 g L -1 , yeast extract: 5 g L -1 , K 2 HPO 4 : 1 g L -1 , agar: 15 g L -1 , Czapek concentrate: 10 mL (Plewa-Tutaj and Lonc, 2014).The cultures were incubated for 5 days at 30°C (as recommended by MTCC).Fungal spores were collected into phosphate buffer saline (PBS) with 0.01% Tween 80 (PBST) and was used as the stock suspension for the inoculation of the test surfaces.The spore concentration of this stock suspension was measured using haemocytometry and maintained at 10 6 spores mL -1 .Under high nutrient condition and submerged culture, fungi grow in the form of vegetative cells rather than spores (Dhingra and Sinclair, 1995).To induce sporulation (formation of fungal spores) on the test surface, a modified nutrient medium with a composition of sucrose-5 g L -1 ; yeast extract-1 g L -1 ; K 2 HPO 4 -1 g L -1 and Czapek concentrate-10 mL, was used.After several trials, this composition, which is relatively less rich in amount of sucrose and yeast extract compared to the fungus culture nutrient, was found to be ideal for sporulation.
Two surface materials, aluminum foil and cardboard, were chosen for the flux experiments.Both these materials are used for packing food and are found in municipal solid wastes.Experiments with aluminum foil were performed by covering the surface of a rectangular glass cavity (75 mm long, 25 mm wide and 2mm deep) with food grade aluminum foil.For the cultivation of fungal spores on cardboard, strips (25 mm wide and 75 mm long) were cut and placed in the flux chamber after sterilization and drying.Different volumes of the sterilized sporulation nutrient CYE were added to the sterilized test surface, up to a maximum of 4 mL along with 20 µL of fungal spore inoculation stock.The inoculated surface was incubated at temperatures between 32-35°C and a relative humidity between 95-99%.The surface source density (number of spores on surface/area) on the test surface was measured at different periods of incubation after inoculation ranging from 1-5 days typically and in some cases up to 14 days.

Measurement of Spores
The impinger fluid (PBST solution) in the Biosampler was used directly for the spore count.Surface spore density estimation for the aluminum foil was done simply by washing the surface using PBST into a collection vessel and quantified.The fungal spores on the cardboard surface were harvested by shaking gently for 30 minutes and the spores separated gently using a vortex mixer.
Fluorescence microscopy was used to measure bioaerosol flux.The liquid suspension containing the spores from the Biosampler were filtered through a black nucleopore filter paper (Whatman ® pore size 0.2 µm; 25 mm diameter) stained by adding 250 µL of a 50 µg mL -1 Acridine orange (Sigma Aldrich ® ) solution and allowed to interact for about 5 minutes in the dark.The dye was expected to complex with the DNA of the fungal spores (both viable and non-viable) and exhibit fluorescence (Palmgren et al., 1986).The stained filter paper was viewed using a fluorescence microscope (Carl Zeiss Axioskop II MAT) with a 50X objective and a 10X eye piece.The images were captured using a digital Camera (Nikon CoolPix 4500).Typically 15-20 images, covering about 0.028 mm 2 of the filter paper were obtained randomly.Each image was processed and the spores were counted using ImageJ software.The total count in the sample was estimated by scaling up this measured count for the area observed to the total area of the filter.Surface spore densities was obtained by using haemocytometry (Dillon et al., 2007).Though this technique cannot differentiate between biological and non-biological aerosols, it was deemed applicable in this work, since these were laboratory experiments and the fungal spores were well known structures.The attraction of using haemocytometry was that it is a much simpler and quicker technique to use compared to fluorescence microscopy.In preliminary studies we observed that the range of spore measurement in a liquid suspension using haemocytometry was between 10 8 -10 4 spores mL -1 .In our studies, this was appropriate for surface spore density measurements since the number of spores were relatively large in these samples.The number of spores in the PBST suspension in the Biosampler used for the flux measurements were in the range of 10 3 -10 4 spores mL -1 , which was observed to be around the lower range of the applicability of haemocytometry in these experiments.Therefore, fluorescence microscopy was seen as a suitable technique for flux measurements, while haemocytometry was used to obtain surface spore density.

Formation of Fungal Spores on Aluminum Foil and Cardboard
The formation of fungal spores was visible to the naked eye as a fairly uniform green color covering the entire test surface 3 days after inoculation (as seen in Fig. S1).
Corresponding to this visual observation on the surface, preliminary flux experiments also indicated that there was no fungal spore release until the 3 rd day after inoculation.Fig. 2 shows the number of spores harvested from aluminum foil and cardboard surfaces on the third day after inoculation.It shows that the surface spore density on aluminum foil increased nearly seven fold from 8.5 ± 4 (× 10 5 ) to 56 ± 7 (× 10 5 ) spores cm -2 as the nutrient dose was increased from 2 mL to 4 mL indicating proportional response to nutrient presence.In the case of cardboard, much higher spore numbers of 257 ± 43 (× 10 5 ) spores cm -2 was observed even with only 2 mL of nutrient addition.Even on a wet cardboard surface (containing 2 mL of deionized water without any CYE nutrient) a spore count of 37 ± 12 (× 10 5 ) spores cm -2 was observed.The cardboard material itself can sustain fungal spore formation without additional external nutrients

Effect of Air Velocity
To investigate the structure of the fungal spores on a surface and the impact of air velocity, microscopic images of the fungal surface was obtained before and after an exposure time of 30 minutes.Figs.3(a) and 3(b) show the side view of the fungal surface before and after exposure to air in aluminum foil respectively.Fig. 3(a) shows the upright hyphae on the surface with the spore-bearing conidia and Fig. 3(b) shows that the hyphae have collapsed.Figs.3(c) and 3(d) show the top view of the spores on aluminum foil before after exposure to air respectively.In Fig. 3(c), before exposure to air, the spores at the end of the upright hyphae are in focus, while the upright hyphae and the background are out of focus implying that the spores are present at a height different.Fig. 3(d) shows the top view after 30 minutes of exposure time and the spores and the hyphae are seen on the ground and in focus together with the surroundings.This is a very significant observation that can be related to the spore release flux due to air exposure.Fig. S2 shows the similar set of images for cardboard.The observations are very similar to that of aluminum foil.
Fig. 4 shows the flux of fungal spores from aluminum surface as a function of the air velocity.Experiments were conducted at five different air velocities -2.0, 4.3, 6.3, 8.0 and 11.1 m s -1 .All these experiments were conducted with spores cultivated for an incubation time of 3 days.Based on the observation in Fig. 2, the spore density on the aluminum foil surface is expected to be at a level of 56 ± 7 (× 10 5 ) spores cm -2 in all these experiments.The surface was subject to an exposure time of 30 minutes to air at a desired constant velocity and the cumulative flux was measured.At an air velocity of 2 m s -1 , there was no measurable flux.The fluxes measured at air velocities of 4.3 m s -1 , 6.3 m s -1 , 8.0 m s -1 and 11.1 m s -1 were 3078 ± 1716, 10498 ± 2740, 15216 ± 7396, 10643 ± 5962 spores cm -2 min -1 respectively.From Fig. 4, it is evident that there is a minimum threshold velocity required to induce aerosolisation and the spore flux increased as velocity increased from 4.3 to 8.0 m s -1 .The flux at 11.1 m s -1 however is similar to the flux obtained at 6.3 m s -1 .The fluxes reported in Fig. 4 also were corrected for bioaerosol sampling losses, which were measured and added to the measurement obtained from the biosampler.The average overall deposition losses as a percentage of the total flux values were found to be 32.6 ± 16.7 % (n = 8).
Based on the structure of spores, shown in Fig. 3, it is hypothesized that a certain amount of energy is required to dislodge the spores from the conidia.This energy supplied by the air is estimated by the shear or drag force at the surface of the spores.The drag force on the spores can be estimated by Eq. ( 2) (Geagea et al., 1997) 2 2 1 8 where ρ a is the density of air, u is the velocity under at the surface of the spores, d p is the spore diameter and C D is the drag coefficient.C D is a function of the spore Reynolds Number, Re P defined as Re P = d p ρ a u/µ a , where µ a is the viscosity of air.Based on this definition, the flux of spores, N may be estimated as where S is the surface spore density (number of spores/ area).The numerator in Eq. ( 3) represents the rate of total energy transferred to the spores as a result of the air movement.The flux therefore is a strong function of the velocity of air at the surface of the spores, which in this case depends on the velocity profile above the surface.The denominator in Eq. ( 3) contains terms that represent the different energies required to dislodge one spore and Exptl.

Model
aerosolize it.The terms E A and E G refers to adhesive and gravitational energies respectively.E A was estimated to be of the order of 10 -19 J, using the relationship Ad p /24d, (Hiemenz and Rajagopalan, 1997) where d is the minimum equilibrium separation distance (assumed to be 10 nm) and A is the Hamaker constant, which is assumed to be that between two carbon particles (Maurer et al., 2001) and of the order of 10 -20 J.The fungal spore diameter was estimated from microscopic measurements to be of an average size of about 3.5 µm.E G is estimated using the relationship E g = GM m /R where G is the gravitational constant, M is the mass of the earth, R is the radius of earth and m is the mass of the spore.Using a spore particle density, ρ p , be 1100 kg m -3 (Kanaani et al., 2008) and spore radius as 3.5 µm.E G is estimated to be of the order of 10 -8 J. Noblin et al.
(2009) report the measurement of a spore rupture force to simulate the active release of basidiomycota, where a surface tension modification triggers spore release.The energy parameter, E P , is an analogous physical breakage energy parameter, that is proposed in this study to represent the energy required to break a spore from the aerial hyphae by a drag force caused by air flow.E P is estimated from a linear fit between the experimental flux (N) and the term (F D .u.S).
The reciprocal of the slope is the sum of the energies (E A + E G + E P ).Fig. 4 shows the model fit (using Eqn3) for the four experimental points (corresponding to air velocities 2, 4.3, 6.3 and 8.0 m s -1 ) shown where the flux increased with velocity and E P , the energy required to break one spore, was estimated as 0.986 mJ.It is clear that E P >> E G + E A , therefore rendering E A and E G insignificant.The term E P is expected to be a function of the fungal spore species and other factors such as relative humidity, temperature or age.
Estimation of E P using other experimental techniques may be attempted to measure the physical breakage force for spores to compare the estimate of this parameter using Eq. ( 3).The model represented by Eq. ( 3), showed an increase in spore release with increase in air velocity.At very low air velocities, when the total energy transferred is smaller than the minimum energy required to dislodge one spore from the conidia and this corresponds to the threshold velocity.Based on Eq. ( 3), the velocity corresponding to a condition where F D. u.S = E P is the threshold velocity.The experimental data at an air velocity of 11.1 m s -1 air is nearly the same as the flux obtained at an air velocity of 6.3 m s -1 suggesting that there is a maximum velocity beyond which the energy transfer does not change.The model does not predict the experimental flux at 11.5 m s -1 .Based on the images in Fig. 3 and the model, it is hypothesized that at velocities higher than a certain upper limit some structural change (bending or collapse) to the fungal hyphae that holds the conidia may result in the shift in the spore position relative to the bottom surface and therefore the velocity in contact with the spore might change resulting in lower drag and corresponding energy transfer.This must be investigated using microscopy to observe the dynamic mechanical response of the hyphae and the spores due to variation in air velocity.This response can then be represented by a more refined version of the Eq.(3).
Fig. 5 shows another set of flux experiments conducted at a constant air velocity of 4.3 m s -1 where the surface was continuously exposed for about 2 hours at 30 minute sampling intervals.The flux was 3842 ± 1356 spores cm -2 min -1 for the first 30 minutes and dropped to a value of 371 ± 101 spore cm -2 min -1 , 52 ± 4 spore cm -2 min -1 and 26 ± 20 spore cm -2 min -1 in 60, 90 and 120 minutes respectively.At this point, the air velocity was increased to 12.9 m s -1 and sampled for 30 minutes.The flux increased to a value of 5484 spores cm -2 min -1 .Based on the model, when the aerial hyphae have collapsed on the surface after exposure to air, the drag force on the spores is different from when the hyphae are upright.The heights of the upright and collapsed hyphae were visually estimated (from images) as 1.0 mm and 0.1 mm respectively.Based on the laminar flow velocity gradient in the momentum boundary layer above the surface in a rectangular channel for an average velocity of 4.3 m s -1 , it is estimated that the velocity near the top of the upright hyphae is 3.82 m s -1 and the velocity near the collapsed hyphae is nearly 0.55 m s -1 .Using these velocities in Eqs. ( 2) and (3), and using the fit parameter E P , the model estimate of the flux is shown in Fig. 5.The corresponding model predicted fluxes were 47 spores cm -2 min -1 for 60, 90 and 120 minutes assuming that the hyphae had collapsed.Fig. 5. Extended exposure time of fungal spores to air at 4.3 m s -1 (surface spores density maintained at 56 ± 7 (× 10 5 ) spores cm -2 ± 11 (× 10 6 ) spores) till 120 minutes followed by an increase of air velocity to 12.9 m s -1 .
When the average velocity was increased to 12.9 m s -1 , the corresponding velocity at 0.1 mm was estimated as 3.94 m s -1 .The predicted flux was 2880 spores cm -2 min -1 and the measured flux was 5400 spores cm -2 min -1 .The difference between the model and experimental flux values at 12.9 m s -1 might be due to other effects that are not considered by the model such as the turbulence regime corrections due to the higher velocity.

Effect of Surface Spore Density
Fig. 6 shows the correlation of bio-aerosols emission flux with the number of spores present on the aluminum foil surface.Flux measurements were made after 30 minutes of exposure time at constant air velocity 4.3 m s -1 .It was observed that the spore flux was higher for larger surface spore density.The x-error bars indicate the standard deviation in the surface spore density analysis (n = 3) and the y-axis error bars indicate the variation in fluxes measured (n = 3).There is clearly a positive correlation between the flux and surface source density.Visual observation of surfaces showed the presence of higher density of aerial hyphae in the form of clusters with higher measured surface spore densities.Fig. 6 also shows comparison with the model with drag terms estimated for upright hyphae and the energy terms estimated earlier but using different average values of S (surface spore density) measured and shown in Fig. 2 in Eq. ( 3).The model predicts the experimental fluxes reasonably well.Surface spore density is a factor that is dependent on the fungal species, nutrient type and other environmental parameters.A-priori estimation of S can be obtained from experimental measurements of spores on the surface or from equations that predict the formation of fungal spores depending on fungal species type, nutrient type and surface morphology.

Effect of Incubation Time and Surface Type
Fig. 7 shows the flux of fungal spores from aluminum and cardboard surfaces as a function of incubation time after inoculation in a single event short term exposure of air at an air velocity of 4.3 m s -1 .The flux of fungal spores from a 3-day old incubated aluminum surface was found to be 3078 ± 1715 spores cm -2 min -1 (n = 4).For surface sample of aluminum that was incubated for longer periods (5, 12 and 20 days) with the same initial volume of nutrient, no flux was observed.Measurements of the surface spore density of 5-day old surfaces showed statistically insignificant changes in surface spore densities as the 3-day sample suggesting that the lack of release is not related to lack of spores on the surface.Flux from cardboard surface was 4168 ± 2643 spores cm -2 min -1 (n = 3) after three days of incubation.The cardboard surface had no external nutrient added except for water to obtain surface spore density similar to that on the aluminum surface at 3 days (based on data shown in Fig. 2).However, it was observed cardboard Flux (spore/cm 2 min)

Incubation Time (days)
Aluminum foil Cardboard surfaces that were incubated for 5 and 12 days showed significantly higher flux than those aged for 3 days.The flux reduced to very low values from surfaces incubated for 20 days.This effect was also observed by Geagea (1997) and Madsen (2006), however, without any specific explanation.
To investigate the reason for the flux reduction, the surface was observed under a microscope.This showed that the aerial hyphae had collapsed after an incubation period of 5 days even without exposure to air.In contrast, surface images of the cardboard surface showed that the aerial hyphae were upright until about 20 days before partially collapsing.When this observation is considered in conjunction with spore flux data, the reduction in flux on the 5 th day on aluminum and on the 20 th day on cardboard surfaces are probably due to the collapse of the aerial hyphae when nutrient is depleted on the surface.It is clear from the aluminum case that there is possibly no nutrient remaining after 3 days.Additional experiments conducted to check the water evaporation from aluminum foil show that the surface contains about 35% of the initial water on the 3 rd day implying that the CYE nutrient is consumed.Expeirments with all the different volumes of CYE solutions show the same effect implying that water is not the controlling factor for the spore formation in this case.For cardboard, the water penetrates the material below the exposed surface and therefore water is available for longer in the sytem and also facilitates the metabolism of the nutrients associated with cardboard to support fungal spore formation.However further studies are required to investigate the exact nature of nutrient depletion and availability in cardboard.It will also be interesting to investigate the dynamics of surface moisture due to absorption and wetting in the nutrient availability especially in the case of cardboard.

CONCLUSIONS
The experimental data and modeling results in this study have given additional insight into the mechanism and the factors affecting wind-induced passive release of fungal spores.The combination of flux measurements and imaging have given rise to the hypothesis that the fungal aerial hyphae collapses under two conditions -a) due to the shear force of the wind and b) when there is nutrient depletion.When the aerial hyphae collapse additional energy is required to aerosolize them thus resulting in a decrease in spore release and therefore requires higher air velocities to maintain spore release.In a real scenario of exposed solid waste, this implies that the longer term flux is low and is likely to increase if there are higher sudden gusts of wind.It was also observed that the kinetics of the fungal growth plays an influential role and depends on the nutrient availability -either from a food source or from a surface that can supply nutrients.The data also suggests that there is no direct effect of the surface on the aerodynamics of spore release since spore are not attached to the surface directly but to a hyphae that rise from the mycelium which grows on the surface.However, the surface type and structure might have an impact on the kinetics and magnitude of spore formation and therefore have an effect on the spore flux.Many aspects of the surface influence on the spore formation must be investigated in detail in independent studies.A preliminary mathematical model presented here using simple energy balance using system and bioaersosol specific factors that influence spore release.Among the different energy barriers, the physical breakage energy parameter, E P was found to be the most significant.E P was estimated for Penicillium chrysogenum from one set of experimental data and was used to predict experimental fluxes for other set of experimental conditions.For the model to be truly predictive, the term E P must be estimated from alternative experimental techniques.If the values are comparable with the experimental fit for a specific species of fungal spores, these flux experiments can be used as a surrogate for estimating E P. Additionally, the term S must also be predictable using kinetic for fungal spore formation using different types of nutrients available in municipal solid waste.

Fig. 2 .
Fig. 2. Surface spore density after 3 days of incubation as a function nutrient dose.(Error bars indicate standard deviation of n = 3).In the case of wet cardboard only 2mL water was added with no CYE nutrient.

Fig. 3 .
Fig. 3. Effect of air flow on fungal spores on Aluminum foil a) 3 rd day before air flow (b) After air flow with an exposure time of 30 minutes(c) 3 rd day before air flow-top view (5X) (d) After air flow-top view (5X).

Fig. 4 .
Fig. 4. Spore flux as a function of air velocity and comparison with model fit.