Annals of Occupational Hygiene Advance Access originally published online on December 14, 2005
Annals of Occupational Hygiene 2006 50(2):175-187; doi:10.1093/annhyg/mei057
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© 2005 British Occupational Hygiene Society Published by Oxford University Press
Original Article |
Characterization of Microbial Particle Release from Biomass and Building Material Surfaces for Inhalation Exposure Risk Assessment
National Institute of Occupational Health, Lersø Parkallé 104, 2100 Copenhagen, Denmark
* Author to whom correspondence should be addressed. Tel: 0045 39 16 52 42; fax: 0045 39 16 52 01; e-mail: amm{at}ami.dk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONACKNOWLEDGEMENTSREFERENCES |
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A conceptual approach including measurements of materials at rest (step 1), measurements using a large rotating drum (step 2) or a Particle-FLEC (step 2) and measurements at a workplace (step 4) has been used to characterize the release of microbial components (bacteria, fungi, actinomycetes, endotoxin or enzymes) and particles from straw, wood chips or fungal cultures of different ages on gypsum boards. Repeated agitation or handling periods were included in step 2 and step 4. There was a low similarity between the amount of microbial components measured in step 1 and the aerosolized amount (step 2) from gypsum boards, wood chips and straw. Ratios between some microbial components measured at the workplace (step 4) and measured in step 2, showed similarities. Less than 1.3% of the total amount of microorganisms and endotoxin becomes airborne during 5 min of agitation of straw or wood chips. Most microbial components were released at higher rates during the first agitation period than during the following periods. However, differences were seen between different microbial components, and endotoxin from straw was released at the same rate in two successive agitation periods. Fungal particles smaller than spores were released from fungal colonized gypsum boards at amounts that were up to 30 times higher in the first agitation period compared with that in the following period, while fungal spores were released at amounts that were five times as high in the first period compared with that in the following period. In addition to differences between microbial components, the release patterns of microbial components were different for wood chips and straw. The time for maximum particle release to half particle release was longer for straw than for wood chips. The observation that some components, e.g. endotoxin, are released at the same rate in two successive handling steps, and that others (e.g. fungi) are mainly released initially, shows that the exposure period to different components from the same material differs in duration. The observed differences in the release patterns of different components and the differences between materials are important when preventive steps are to be taken, and it stresses the importance of applying a relevant sampling time and period in exposure assessments.
Keywords: actinomycetes • bioaerosol • endotoxin • enzymes • fungi • dustiness • exposure • straw
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONACKNOWLEDGEMENTSREFERENCES |
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Respiratory health problems have been associated with occupational exposure to particles of microbial origin during handling of organic materials such as wood chips, straw, grains, hay and household waste (Thörnqvist and Lundström, 1982
; Malmberg et al., 1985; van Assendelft et al., 1985; Jäppinen et al., 1987; Kolmodin-Hedman et al., 1987; Sigsgaard et al., 1994; Eduard et al., 2001; Melbostad and Eduard, 2001; Viet et al., 2001). Studies in environments where materials containing fungal enzymes are handled indicate that many enzymes can cause respiratory disorders (e.g. Houba et al., 1996; Vanhanen et al., 1997, 2001; Cullinan et al., 2001). In indoor environments current associations between mould growth on building material surfaces and increased prevalence of airway irritation, allergy and asthma are equivocal (Bornehag et al., 2001). Methods for measuring indoor bioaerosol exposure and health assessment are not well standardized, making interpretation of existing data difficult (Fung and Hughson, 2003). Exposure assessment for epidemiology and for problem identification in specific work scenarios and buildings is hampered by insufficient knowledge of the complex behaviour of microbial growth and particle release. Experimental approaches are needed for gaining insight into parameters governing microbial growth and particle release behaviour, including emission rates. Such approaches are also useful for the initial assessment of exposure risk in specific scenarios, e.g. handling of bulk materials with varying extent of mould growth, or assessment of exposure risk from a visible patch of mould growth in a building.
Table 1 proposes a conceptual approach to the characterization of microbial particle release for research and for risk assessment in practice. The approach involves steps of increasing complexity and of increasing similarity with real exposure scenarios. Taking biomass, e.g. straw, as an example, step 1 consists of the analysis of a well-defined measure of the total amount of specific microbial components per weight of bulk material. Methods such as quantitative extraction from a sample, e.g. by washing and subsequent analysis (Hayes, 1969; Amner et al., 1988; Magnoli et al., 1998), or by cutting the studied material into small segments and placing it on a agar medium (Willcock and Magan, 2001) could be used. The results can be used for hazard identification.
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Step 2 characterizes the propensity for airborne particle release. It consists of a bench-scale test that agitates the materials using a well-defined challenge.
Several methods for use in step 2 have been described in the literature, which simulate different handling scenarios. One method is the use of a rotating drum. This method simulates repeated dropping of the material from a given height. A small (0.3 m internal diameter) rotating drum was used by (Breum et al., 2003) to characterize the release of dust, fibres and endotoxin from organic insulation materials. The results showed that the method had potential for ranking dustiness in a similar order as the measured dust exposure risk of persons installing the material. For very inhomogeneous materials such as straw, wood chips and household waste, a large (3.3 m3) rotating drum has been used for studying release of microorganisms, endotoxin and dust (Breum et al., 1997, 1999; Nielsen et al., 1997; Madsen et al., 2004). In power plants using biofuels such as straw and wood chips, worker exposure mainly occurs during manual or mechanical handling of the bulk material. Thus, the rotating drum dustiness tester is well suited for studying factors that influence release of airborne particles and, hence, exposure of workers at these workplaces. It has been shown that release of dust particles varies with time and that it can either decrease or increase as the agitation goes on (Madsen et al., 2004). This implies that materials differ with regard to the initial content of dust and particle release due to attrition. This has important consequences for assessing exposure risk for work scenarios involving one short, repeated short or prolonged agitation of biofuels, and how to include results of dustiness tests in such risk assessments.
In indoor environments, the main causes of microbial particle release from building surfaces are air currents (Harney et al., 2000) and building vibrations (Harney et al., 2000). Methods for step 2 could include air jets (Górny et al., 2001; Kildesø et al., 2003) and vibration (Harney et al., 2000). Kildesø et al. (2003) used a specially designed instrument P-FLEC (Particle-Field and Laboratory Emission Cell), which agitates the surface by a row of air jets. The jets are scanned over a well-defined surface area, and the particles generated are collected and quantified. The jet velocity and the scanning velocity can be adjusted to simulate different agitation scenarios. Using this method, Kildesø et al. (2003) found that spore release was dependent on species and air velocity.
Step 3 consists of full-scale simulation, and the ultimate step, step 4, would be a complete assessment of personal exposures.
The aim of this study has been to characterize microbial particle release from straw, wood chips and mouldy gypsum boards using the conceptual approach (Table 1). The specific objectives were to:
- Compare results obtained in step 1 and step 2 for straw, wood chips and gypsum board surfaces.
- Study the effect of repeated agitation in the rotating drum (steps 2.1 and 2.2) of straw and wood chips on the release of airborne actinomycetes, different fungal species, bacteria, endotoxin, enzymes and particles in general.
- Study the effect of agitation by repeated air jets (steps 2.1, 2.2 and 2.3) on the release of Penicillium chrysogenum and Trichoderma harzianum spores from gypsum board surfaces.
- Compare results obtained in step 1 and step 2 for straw with results obtained by sampling aerosols at a biofuel plant (step 4).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONACKNOWLEDGEMENTSREFERENCES |
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Biomass for agitation by the rotating drum
Approximately 5 kg of material was sampled from each of the 18 different piles of straw from wheat (Triticum aestivum) and 12 different piles of wood chips from sour felled Norway spruce (Picea abies). The samples were stored in open black polyethylene bags at 9–15°C for up to 24 h. The straw and wood chips were between 0 and 14 months old, but the influence of biomass age was not studied. The water content ranged from 11 to 13% (w/w) for the straw and from 32 to 40% (w/w) for the wood chip samples.
Cultivation on building materials for P-FLEC measurements
T. harzianum (IBT 9153) and P. chrysogenum (IBT 14920) were cultivated on pieces (0.08 m2) of sterilized wet wallpapered gypsum boards and incubated at a relative humidity (RH) of 
95% as described in the following. Distilled milli-Q water was used for wetting the boards. Inoculation was performed by spraying a spore suspension (106 spores/ml) onto the boards (1 x 107 spores/m2) using an atomizer. The gypsum boards were placed in stainless steel boxes with tightly fitting glass covers. A saturated solution of potassium sulphate controlled the RH. The fungi were incubated at 22°C in darkness for 8 days to 6 weeks.
The two fungi were chosen for this study as they are commonly found in many environments including mouldy buildings and soil, and furthermore, as T. harzianum, in contrast to P. chrysogenum, produces its spores in slimy masses, which may have an effect on the spore release.
Total amount of microbial components in biomass (step 1)
A sub-sample of 100 g was sampled from each of the 18 straw and 12 wood chip samples and shaken (250 r.p.m.) in isotonic water for 15 min on ice. This procedure is called washing (step 1). The pH ranged from 6.2 to 7.0 for the straw suspensions and from 4.5 to 5.0 for the woodchip suspensions. The suspensions were used for counting the total number of fungi and bacteria, for colony forming units (cfu) of fungi and bacteria and for endotoxin analysis (Table 1).
Total amount of spores on building material surfaces (step 1)
In the experiments with release of particles from fungal cultures of different ages, two areas each of 13.2 cm2 of the wallpaper were cut from areas of the gypsum boards that had not been agitated by air jets. The colonized wallpaper was shaken (orbital, 500 r.p.m., 15 min) in a sterile 0.05% Tween 80 and 0.85% NaCl aqueous solution, following this the total number of spores was counted using a haemocytometer. The experiments were performed in triplicate (Table 1).
Agitation of biomass by the rotating drum (step 2)
A rotating drum with horizontal axis and a volume of 3.3 m3 as described by Breum et al. (1999) was used to agitate biomass. The bulk material (3 kg) was placed into the bottom of the drum, which was then rotated at 7 r.p.m., first, for 5 min and, then, for 10 min. An isokinetic probe downstream of the drum delivered a sub-sample (1.9 l min–1) to a particle counter (GRIMM model 1200) measuring particle concentrations over 6 s intervals. Dust for microbiological analysis was sampled during the first 5 min (step 2.1) and again during the following 10 min (step 2.2) of agitation on three filter cassettes located 0.2 m upstream from the outlet. One cassette contained a Teflon filter in closed-face filter cassettes (25 mm diameter, pore size 3 µm; Millipore membrane filters, Ireland) with a 5.6 mm inlet at an airflow of 1.9 l min–1, and two cassettes containing polycarbonate filters in closed-face filter cassettes (25 mm diameter, pore size 0.4 µm, Nuclepore, Cambridge, MA) with a 4.4 mm inlet at an airflow of 1.9 l min–1. All remaining particles were sampled downstream of the probe on a 140 mm diameter 8 µm cellulose nitrate membrane filter (Sartorius, Göttingen, Germany) using a vacuum pump. This maintained a total airflow of 420 l min–1 through the drum. HEPA filtered replacement air was supplied upstream of the drum and in excess ensuring ambient pressure inside the drum.
The mass of dust collected on Teflon filters and cellulose nitrate filters was determined by weighing the filters before and after sampling. Before weighing, the filters were equilibrated at a constant air temperature and humidity for 24 h. The Teflon filters were frozen at –80°C and later used for measuring the endotoxin and enzyme content. The two polycarbonate filters were pooled and used for determining the number of microorganisms by the CAMNEA method as described later.
P-FLEC measurements (step 2)
The P-FLEC (Chematec, Denmark) was used for measuring the release of airborne particles from building material surfaces as described by Kildesø et al. (2003). The air jets were directed towards the surface at an angle of 45° and with a mean velocity at the surface of 1.5 m s–1. The jets were scanned over the surface covering a surface area of 130 cm2. The duration of one complete rotation was 60 s and each measurement lasted 60 s. The particles were transported by the airflow to the outlet at the top. Particles were either measured with an Aerodynamic Particle Sizer (APS 3320, TSI Inc.) in the size range 0.5–20 µm, or collected on a polycarbonate filter (pore size 0.4 µm) and the spores counted in a haemocytometer.
In one experiment the particle release was studied to see how it was affected by repeated agitation by air jets of exactly the same area; this experiment was repeated on four gypsum boards for each fungus. In another experiment the particle release from cultures of different ages was studied; with three separate samples for each incubation time.
Sampling of bioaerosols at a biofuel plant (step 4)
Inhalable bioaerosols were sampled using stationary GSP (CIS) inhalable samplers for 6 h in two working areas, namely ‘straw reception’ and ‘straw shredder’ at a Danish biofuel plant. Straw reception is the first handling period of straw at the plant (step 4.1). Next the straw is transported at the plant and then cut into pieces by a straw shredder (step 4.2). The samplers were mounted with Teflon filters (pore size 1.0 µm) for endotoxin analysis and polycarbonate filters (pore size 1.0 µm) for quantification of total number and cfu of bacteria and fungi (methods described later). The straw handled at the plant had a water content (w/w) of average 15% (min = 12%, max = 23%) and 115,300 kg of straw was received at the plant on the two days.
Determination of endotoxin by the Limulus method
Dust was extracted with 5.0 ml sterile 0.05% Tween 20 aqueous solution by orbital shaking (300 r.p.m.) at room temperature for 60 min and centrifuged (1000x g) for 15 min. The supernatant was analysed (in duplicate) for endotoxin by the kinetic Limulus Amboecyte Lysate test (Kinetic-QCL endotoxin kit, BioWhittaker, Walkersville, MD). A standard curve obtained from an Escherichia coli O55:B5 reference endotoxin was used to determine the concentrations in terms of endotoxin units (EUs) (14.0 EU 
1 ng). The total amount of endotoxin in EU released during the agitation period per kilogramme of biomass per minute of agitation or per milligramme of dust was calculated.
Quantification of microorganisms from biomass dust (CAMNEA)
Microorganisms were quantified by a modified CAMNEA method (Palmgren et al., 1986). A sterile 0.05% Tween 80 and 0.85% NaCl aqueous solution was added to a filter cassette (within 2 h after dust sampling), which was then shaken (500 r.p.m.; 15 min). The number of cultivable fungi was counted by plating dilutions on DG 18 agar (Oxoid, Basingstoke, England) followed by incubation at 25°C. The number of bacteria (25°C), mesophilic actinomycetes (25°C) and thermophilic actinomyctes (55°C) culturable on Nutrient agar (Oxoid, Basingstoke, England) with cycloheximide (50 mg l–1) was also measured. Data for mesophilic and thermophilic actinomyctes were pooled. The number of cultivable fungi, actinomyctes and bacteria per milligramme released biomass dust was calculated and are abbreviated as Cfungi, Cactinomyctes and Cbacteria. In addition the number of released cultivable microorganisms per kilogramme biomass per minute (average release rate) was calculated.
The total number of bacteria and fungal spores was determined after staining in 20 p.p.m. acridine orange (Merck) in acetate buffer. Fungi and bacteria were counted by microscopy (Orthoplan; Leitz Wetzlar). The number of microorganisms was determined in 40 randomly chosen fields or until a total number of at least 400 spores were counted. The total number of fungi and bacteria per milligramme released biomass dust are abbreviated as Tfungi and Tbacteria.. In addition, the number of released total microorganisms per kilogramme biomass per minute was calculated.
Quantification of enzymes
To quantify the activities of NAGase (EC3.2.1.30) (at 50°C), the cellulase ß-glucosidase (EC 3.2.1.21
[EC]
) (at 37°C), 
-amylase (EC 3.2.1.1
[EC]
) (at 37°C) and xylanase (EC 3.2.1.37
[EC]
) (at 50°C) release of p-nitrophenol from p-nitrophenol-N-acetyl-ß-D-glucosaminide; p-nitro-phenol-N-acetyl-ß-D-glucopyranoside; 2-chloro-4-nitrophenyl-ß-D-maltoheptaoside or p-nitrophenyl--D-xylopyranoside (Sigma Chemical Co., USA) was quantified according to Madsen (2003) and Lama et al. (2004). Appropriate controls without either the enzyme or the substrate were run simultaneously. One unit of enzyme activity is defined as the amount of enzyme, which releases 1 µmol of p-nitrophenol ml–1 enzyme min–1. The enzyme activities per kilogramme biomass per minute of agitation (average release rate in the agitation period) were calculated.
Release rate and concentrations of microbial components in biomass dust
The dustiness index, denoted DX, is defined as the amount of airborne particle mass or microbial component, X, generated per mass of tested material, MBIOMASS (Breum et al., 1999). In the present study, particle mass and components, X, were determined in the air samples collected on the teflon or polycarbonate filters and the release rate was calculated from the equations:
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The particle generation rate, S, can be calculated from the concentration time series, Ci, by the equation (Hjemsted and Schneider, 1996):
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t is the time between two consecutive measurements, and 
= V/Q = 472 s is the time constant of the drum (Q = total airflow rate). It is assumed that t << .
Statistical analysis
Statistical analyses were performed with SAS (version 8e, SAS Institute, Cary, NC). The microbial dustiness of biofuels was log-normally distributed and was thus log-transformed prior to statistical analysis. The P-FLEC results were normally distributed. To compare the amounts of each microbial component released during the first and second Agitation, PROC GENMOD (GENeralized linear MODels, distribution = binomial) was applied using the following model in SAS specified as: RX/W = Tagitation. W is the amount of microbial componentx released by washing. For step 2.1 this is the amount released in step 1, and for step 2.2 this is: (the amount released in step 1) – (the amount released in step 2.1).
MINITAB (version 14) was used to construct dendrograms with single linkage correlation coefficient distances for release rates obtained in step 1, step 2.1 and step 2.2. These dendrograms are measures of how closely the releases in the different steps resemble each other. Similar cases are joined by links whose position in the diagram is determined by the level of similarity, s(i, j). The distance between two clusters i and j is the minimum distance between a variable in one cluster and a variable in the other cluster. The distance measure, dij, is defined as
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To compare results obtained in steps 1 and 2 with results obtained in step 4, ratios between microbial components released during step 1, step 2.1, step 2.2, step 4.1 and step 4.2 were calculated and compared using PROC GENMOD.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONACKNOWLEDGEMENTSREFERENCES |
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Comparison of measurements in step 1 and step 2.1
The total amount of microbial components obtained by washing (step 1) and the aerosolized amount obtained by agitation of the biomass by the rotating drum (step 2.1) were compared by (i) calculating the median of the ratios between results from step 2.1 and step 1 and (ii) calculating the correlation between the results from step 2.1 and step 1. It is seen that only a small and variable fraction of the total microbial components were released in step 2.1 and only significant correlations between the results obtained in step 1 and step 2.1 were found for cultivable bacteria (Table 2).
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When agitated by air jets (step 2), colonies of T. harzianum and P. chrysogenum on gypsum boards released only small fractions of the total number of spores quantified in step 1. P. chrysogenum released a higher fraction of its produced spores than T. harzianum (Fig. 1) and the release of P. chrysogenum spores in step 2 was age dependent. The particle size distribution indicated that airborne T. harzianum mainly occurred as agglomerates of conidia with aerodynamic diameters larger than spore size (>3 µm), while P. chrysogenum mainly occurred as single spores with an aerodynamic diameter between 2.6 and 2.8 µm. The number of spores released (step 2) did not correlate with the total number of T. harzianum spores (r = 0.60, P = 0.34) and P. chrysogenum spores (r = 0.34, P = 0.14) (step 1).
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Release of microbial components from biomass during repeated agitation (step 2.1 and 2.2)
Results were obtained for each biomass sample after a 5 min agitation period (step 2.1) and a subsequent 10 min period (step 2.2). The total number and cfu of fungi and bacteria and endotoxin, the enzymes NAGase and ß-glucosidase and numbers and mass of particles were above the detection limit in all dust samples. The enzymes xylanase and
-amylase were detected in, respectively, 22 and 24 of the 60 dust samples, and actinomyctes were found in 58 of the 60 dust samples. The release rates of cfu of fungi, bacteria and actinomycetes, the total number of fungi and bacteria, and the amount of endotoxin during steps 2.1 and 2.2 relative to the total amount measured in step 1 were compared. The relative release rates of all components were significantly different in the two agitation periods (P < 0.0001, two sided). The release rates during step 2.1 were higher than during step 2.2 for most components, and this was most pronounced for wood chips (Fig. 2). The dust released during step 2.2 had a lower content of most microbial components (microbial component per milligramme dust), but a higher content of some enzymes (enzyme per milligramme dust) (data not shown).
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For five of the microbial components, the similarities between the release in steps 1, 2.1 and 2.2 are illustrated in dendrograms (Fig. 3). The dendrograms show a high similarity between the release of endotoxin in step 2.1 and step 2.2 from both straw and wood chips, and a much lower similarity between the release in step 2.1 and step 2.2 of fungi from wood chips and of cfu bacteria from straw.
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The time series for particle release of straw and wood chips showed a decrease. The time for maximum particle release to half particle release was longer for straw than for wood chips (Fig. 4).
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Comparison of measurements in steps 1, 2.1, 2.2 and steps 4.1 and 4.2 for biomass
The similarities between the total amount of the different microbial components (step 1) and the amount released during repeated rotation periods (steps 2.1 and 2.2) are illustrated in dendrograms (Fig. 3). The dendrograms show a lower similarity between step 1 and step 2.1 than between measurements in step 2.1 and step 2.2.
Ratios between cultivable fungi and bacteria and between cultivable actinomycetes and bacteria were lower during step 2.2 than during step 2.1. The two ratios mentioned also were lower in a straw shedder area (step 4.2) than in an area where the straw arrives at the plant (step 4.1) (Table 3). Furthermore, ratios between the total number of bacteria and fungi released from straw were at the same levels at the straw plant as in the rotating drum.
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Release of particles from gypsum boards during repeated agitation by air jets
The air jets were scanned three times over the same surface. Size-resolved particle concentrations during the first, second and third agitation by air jets (steps 2.1, 2.2 and 2.3) showed that significantly more spores and microparticles (particles smaller than spore size) were released during the first agitation than during the following agitations (Fig. 5). Microparticles were released at amounts that were up to 30 times higher in the first agitation period compared with that in the following period.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONACKNOWLEDGEMENTSREFERENCES |
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In this study we have used a conceptual approach to characterize the release of microbial components and other particles from biomass and infested gypsum boards. Step 1 seems not to be a good predictor of the release of microbial components from a material because it was observed that (i) there is a low similarity between the amount of microbial components released in step 1 and the amount in step 2, (ii) more specifically ratios between the number of released specific micoorganisms are not conserved going from step 1 to step 2 (Table 3) and (iii) the release in step 2 is dependent on the maturity (age) of the culture (gypsum boards) (Fig. 1b), and on the growth medium (straw or wood chips) (Fig. 2; Table 2). In addition, it is only practicable to use relatively small samples in step 1 in contrast to those in step 2. This makes it difficult to obtain representative samples for step 1. An advantage of step 1 is, however, that it does not demand any special laboratory equipment.
The results obtained by the rotating drum (step 2) and by the exposure measurements at the workplace (step 4) showed the same tendency regarding the ratio Tfungi to Tbacteria. Furthermore, the results were in agreement concerning the temporal dependence of the ratio between Cactinomycetes and Cbacteria and between Cfungi and Cbacteria (Table 3). This comparison could only be made for straw and not wood chips since it was not possible to identify two successive handling steps of wood chips at the biofuel plants. At the biofuel plant, the ratio of endotoxin to Tbacteria during the second handling period did not differ from the ratio found during the first handling period. This is in contrast to the ratios obtained by the rotating drum in step 2.1. and step 2.2 (Table 3). The ratio of endotoxin to Tbacteria during step 2.2 was at the same level as found at the biofuel plant. The ratio results suggest that the rotating drum can mirror release mechanisms at the workplace. No direct comparison was made of the results obtained by the P-FLEC and by measurements in mouldy buildings.
Tests at the step 2 level have given some interesting quantitative data on the release of dust and microbial components showing complex differences between microbial components. For example, cultures of P. chrysogenum on gypsum boards released a fraction of its spores (maximum 4%) two orders of magnitude higher than T. harzianum (maximum 0.03%) (Fig. 1). The low release of T. harzianum may be because T. harzianum in contrast to P. chrysogenum produces its conidia in slime. Differences were also seen between different fungal species concerning the relative release rate in repeated agitations. For example, the release rate of cfu of Penicillium species and T. harzianum from wood chips were, respectively, 16 and 4 times higher during step 2.1 than during step 2.2. During agitation by air jets, 1.6 and 4.8 times more particles were released from cultures of, respectively, P. chrysogenum and T. harzianum during step 2.1 than during step 2.2. A study of spore release from fungal inoculated agar and ceiling boards showed differences between three studied species in the fraction of spores released during the first minutes of exposure relative to the ‘total release’ during 30 min of agitation by air jets (Górny et al., 2001). The variable source strength of P. chrysogenum and T. harzianum on building materials could in part explain why no associations have been found between symptoms and visible dampness or between visible dampness and number of mould spores in the air (Smedje et al., 1997; Ren et al., 2001).
Fungi were in our study released in higher amounts from biomass during step 2.1 than during step 2.2 (Fig. 2). This was also the case for the enzyme NAGase, which is related to the number of fungal spores (Madsen, 2003), and, as for release of fungi, this difference between step 1 and step 2 was most pronounced for wood chips. In contrast, cellulase, which may originate both from the biomass itself and from microorganisms, was released in higher amounts during step 2.2. Xylanase and -amylase involved in the hydrolysis of hemicellulose and starch, respectively, were found only in some samples rendering interpretation based on these findings difficult.
The fungi-bacteria and actinomycete-bacteria ratios were higher during step 2.1 than during step 2.2 (Table 3) and a smaller fraction of the respectively, cultivable bacteria and total bacteria than of respectively cultivable fungi and total fungi were released in step 2.1 (Table 2). This indicates that biomass has a potential to release bacteria during a longer period than to release fungi and, furthermore, that fungi are released more easily than bacteria. Thus the exposure period when handling a material is different for different microbial components. In another study, the actinomycete Streptomyces albus growing on agar released spores as a result of exposure to a very gentle air current of 0.3 m s–1 (Górny et al., 2003), showing that spores of this actinomycete can easily be released.
The fraction of cultivable bacteria released in step 2 was lower than the fraction of the total number of bacteria rendered airborne during agitation of biomass. This may indicate that some bacteria die during aerosolization or sampling. However, the ratio of step 2.1 to step 2.2 was closer to 1 for cfu bacteria than for total bacteria, indicating that many bacteria survive prolonged sampling. Therefore, it is likely that cultivable bacteria are released more slowly than non-cultivable bacteria. This may be because the cultivable bacteria, in contrast to dead bacteria, are attached to the biomass by pili or were present as a bacterial biofilm, which helps the bacteria to resist physical removal during agitation.
P. chrysogenum and T. harzianum released particles that were smaller in size than spores (microparticles) (Fig. 5). This phenomenon was first described in 1999 by Kildesø et al. (1999), and later Green et al. (2005) have described the presence of fragmented conidia in dust collected and cultivated on filters. These microparticles are of special interest in relation to effects on the respiratory system. Microparticles are described as being produced and released by many fungal species growing on different materials (Madsen et al., 2003) and they contain protein (Madsen et al., 2005). In the present study, it was found that microparticles were easily released and that up to 30 times more microparticles were released during the first minute of agitation by air jets than during the next minute. This ease of release of microparticles emphasizes the importance of including microparticles in studies of exposure to fungi.
The tests have shown that the release of actinomycetes, fungal species, enzyme and bacteria is affected by type of material, duration of agitation and age of culture. Thus these factors should if possible be included in any future exposure assessments. The sampling periods should be chosen so that the exposure data could be generalized. The conceptual approach can also provide useful data for developing improved transport, storage and handling methods of biofuels with the aim to reduce exposure to dust and microbial components during use of biofuels and to reduce the generation and transport of bioaerosols to neighbouring areas. Wood chips were found to release about one order of magnitude more total microorganisms, endotoxin and particles (da<5 µm) per second during step 2.1 than during step 2.2 (Fig. 2). The particle generation rate time series showed that wood chips release most of the microorganisms within the first minutes of agitation. This makes exposure during handling of wood chips most pronounced within the first minutes of handling. On the other hand, straw can release bacteria and endotoxin at a high rate for a considerably longer time. Fungal colonized gypsum boards released between 1.5 and 4.4 times more spores during the first air jet exposure than during the following, but an appreciable number was still released during the third air jet exposure. Therefore a fungal-contaminated area of a building material can be expected to release spores during several agitation episodes such as building vibration or air currents. The observation that some species release much higher amounts of spores during the first air exposure than during the following may explain the large variation during the course of a day in the concentration of airborne microorganisms seen in other studies e.g. in Hyvarinen et al. (2001).
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CONCLUSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONACKNOWLEDGEMENTSREFERENCES |
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The proposed conceptual approach was useful for structuring studies of microbial particle release. There was only a low similarity between step 1 (washing of a material) and the aerozolised amount (step 2). Step 1 can be used for hazard identification but was not a good predictor of microbial particle release. Furthermore, <1.3% of the total microorganisms and endotoxin become airborne during 5 min of agitation by a rotating drum of straw or wood chips. The release of fungal spores from gypsum boards (step 2) was dependent on the culture age.
Ratios between microbial components measured at the workplace (step 4) and measured by the rotating drum in step 2 showed similarities, suggesting that the rotating drum is a good method of estimating potential aerosolization of microbial components from straw.
Exposure to total microorganisms, endotoxin, and particles (da < 5 µm) during handling of wood chips is likely to be most pronounced within the first minutes of handling, while straw is likely to release bacteria and endotoxin at a high rate for a considerably longer time. Exposure periods to different components from the same material differ in duration. For example, the period of exposure to endotoxin is likely to be longer than the period of exposure to actinomycetes.
A fungal contaminated area of a building material can be expected to release spores during several agitation episodes such as building vibration or air currents, and the release varies in amount.
The observed differences in the release patterns of different microbial components and fungal species and the difference between materials are important factors to consider when preventive steps are to be taken. The differences, furthermore, stress the importance of observing a relevant sampling time and period in exposure assessments.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONACKNOWLEDGEMENTSREFERENCES |
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Signe H. Nielsen, Tina Trankær Olsen, Mirella Simkus and Gitte Holm are acknowledged for technical assistance. PSO Research Foundation and the Danish Working Environment Council (ASC) are gratefully acknowledged for financial support.
Received June 28, 2005; in final form September 12, 2005
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