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Development and Validation of a Simple Bioaerosol Collection Filter System Using a Conventional Vacuum Cleaner for Sampling

Development and Validation of a Simple Bioaerosol Collection Filter System Using a Conventional... Although numerous bioaerosol samplers for counting and identifying airborne microorganisms are available, the consid- erably high purchase and maintenance costs for the sampler often prevent broad monitoring campaigns for occupational or environmental surveillance of bioaerosols. We present here a newly developed simple adapter and filter system (TOP filter system) designed to collect bioaerosol particles from a defined air volume using conventional vacuum cleaners as air pumps. We characterized the physical properties of the system using air flow measurements and validated the biological performance. The culture-based detection capacities for airborne fungal species were compared to a standard impaction sampler (MAS-100 NT) under controlled conditions in a bioaerosol chamber (using Trichoderma spores as the test organ- ism) as well as in the field. In the chamber, an overall equivalent detection capacity between all tested filters was recorded, although a significant underrepresentation of the TOP filter system for Trichoderma spores were seen in comparison to the MAS-100 NT. In a comparative field study (n = 345), the system showed similar biological sampling efficiencies compared to the MAS-100 NT impactor, only the diversity of identified fungal communities was slightly lower on the filters. Thus, the system is suitable for large-scale environmental sampling operations where many samples have to be taken in parallel at a given time at distant locations. This system would allow endeavors such as antibiotics resistance monitoring or hygiene surveys in agricultural or occupational settings. Keywords Bioaerosol sampling · Air filtration · Sampling device validation · Field study · Bioaerosol chamber · Indoor mold assessment Abbreviations MAS MAS-100 NT (Microbial Air Sampler) bdl Below detection limit MCE Mixed cellulose esters CFU Colony forming unit MEA Malt extract agar MCE Mixed cellulose ester MEA-BR Malt extract agar containing Rose Bengal DG18 Dichloran glycerol agarPTFE Polytetrafluoroethylene (TF) RCS-T Modified HYCON-YM medium sd Standard deviation * Sabine Strauss-Goller VAC Air filtration by filter holder and vacuum sabine.goller@boku.ac.at cleaner Fungal Genetics and Genomics Laboratory, Department of Applied Genetics and Cell Biology, Institute of Microbial Genetics, BOKU-University of Natural Resources and Life 1 Introduction Sciences, Tulln, Austria Center for Health and Bioresources, AIT-Austrian Institute Modern life in industrialized countries dictate that people of Technology GmbH, Tulln, Austria usually spend more than 80% of their daily time indoors Department of Material Sciences and Process Engineering, (Bernstein et al. 2008; Klepeis et al. 2001; Normand et al. Institute of Chemical and Energy Engineering, 2015). Hence, healthy indoor air and monitoring indoor air BOKU-University of Natural Resources and Life Sciences, quality is of great importance and public concern. Exposure Vienna, Austria to microbial air pollutants present as bioaerosols has been AQA Umweltanalytik GmbH, Klosterneuburg, Austria Vol:.(1234567890) 1 3 Aerosol Science and Engineering (2021) 5:404–418 405 related to a wide range of diseases such as allergy, asthma types include membrane filters composed of gelatin (Wu or respiratory disorder (Dannemiller et al. 2016; Madsen et al. 2010), polymer layers and fibrous filters consisting of et al. 2016; Nevalainen et al. 2015; Osborne et al. 2015). cellulose, glass or variant copolymers (Burton et al. 2007; Increased awareness of such potential adverse health effects Haig et al. 2016; Miaskiewicz-Peska and Lebkowska 2012; caused by airborne microorganisms raised the interest in Raynor et al. 2011). Compared to impactors, filtration-based reliable methods for the characterization and collection of samplers cover a wider range of particle sizes with high col- bioaerosols. In addition, fungi and bacteria are prominent lection efficiency depending on the pore size of the mem- pathogens and monitoring of these microbes in aerosols and brane (Aizenberg et al. 2000). One advantage of air filtra- testing their antibiotic or antimycotic resistance profiles is tion is the compatibility with various analytical techniques an important task in public health management (Jones and including cultivation, DNA-based, biochemical, immuno- Brosseau 2015). chemical, and gravimetrical analyses. Generally, high vol- Besides air filtration-based sampling, where airborne ume sampling is desired but particularly for a culture-based particles are drawn through a filter holder and captured on analysis, long-term air filtration may also lead to overloading filters, impaction and impingement are among the most fre- and extended sampling stress (Eduard and Heederik 1998; quently used and well-established air sampling techniques Wang et al. 2001). (Ghosh et al. 2015). Mostly, microbial analysis of bioaero- Besides validation of the physical collection efficiency sol loads relies on cultivation-based techniques that provide according to directives such as DIN ISO 13205-1 (2014), an estimation of culturable microorganisms expressed as air samplers are often compared in field studies to charac- “colony forming units” (CFUs) (Chang et al. 1994; Ghosh terize their overall performance (An et al. 2004; Frankel et al. 2015). Although culturable and non-culturable micro- et al. 2012b; Wang et al. 2015). Discrepancies between such organisms are usually present in bioaerosols international comparative measurements occur due to the generally low standards for the detection and enumeration of these organ- microbial particle concentrations in the environment, parti- isms usually rely on CFU counting and species determina- cle agglomerations, and the unequal distribution of bioaero- tion based on morphological traits. If using a culture-based sols in the air (Fierer et al. 2008; Ghosh et al. 2015; Nazaroff analysis, it has to be considered that an accurate enumeration 2004, 2016). Specific technical limitations of each sampling of the collected airborne microorganisms strongly depends device might amplify these differences. To compensate for on parameters such as cultivability and viability of the such uncertainties, various bioaerosol chambers have been microbial species and the selection of an appropriate culti- implemented to enable stabilized sampling condition (Dyb- vation medium and growth conditions, sampling method and wad et al. 2014; Estill et al. 2008; Feather and Chen 2003; analytical strategy. Furthermore, environmental factors such Kesavan and Sagripanti 2015; Pogner et al. 2019; Simon and as relative humidity, UV radiation, and temperature show a Duquenne 2013; Wang et al. 2001). significant impact on the survival rate of the microorganisms In the present study, we validated a novel filter-based and, therefore, influence a cultivation-based overall assess- sampling system that uses conventional vacuum cleaners as ment (Juozaitis et al. 1994; Tseng and Li 2005; Zhen et al. air pumps. The initiative to develop such a system comes 2018). For air filtration-based sampling, desiccation effects from the necessity of parallel air samplings in large sam- are likely to impair the survival of collected microorgan- ple numbers at many different locations in a short period of isms and might lead to an underestimation of the microbial time. Typically, such sampling campaigns are performed in particles present, particularly of the sensitive ones, such as hygiene surveys or when antibiotic resistance profiles need vegetative cells. (Ghosh et al. 2015; Haig et al. 2016; Wang to be determined over a wide geographic area at a given et al. 2001). Spore forming bacteria and fungi, which are time point. For this, many samplers need to be employed at highly resistant to environmental stresses, are more ame- each of the distant locations and it is financially not feasible nable to monitoring with filtration sampling. Only recently, to equip each of the operators with one of the costly com- molecular methods such as DNA-based quantification mercial bioaerosol samplers. The system described here is of microbes or quantitative biochemical assays (Kespohl low-cost and easy to handle and would thus be well suited et al. 2013; Zahradnik and Raulf 2019) and fungal-specific for such large sampling campaigns. enzyme assays (Reeslev et al. 2003) are starting to be com- monly used for routine measurements and will be imple- mented in ISO Standards worldwide (Rylander et al. 2010; 2 Materials and Methods Unterwurzacher et al. 2018). Air filtration is usually easy to accomplish and depend- 2.1 Description of the Adaptor and Filter System ing on the reason for sampling, a broad spectrum of filter materials featuring different characteristics and pore sizes The system consists of a cylindrical adaptor with a conical are available (Deacon et al. 2009). Commonly used filter inner wall (see Fig. 1A) that is designed to place the adaptor 1 3 406 Aerosol Science and Engineering (2021) 5:404–418 Fig. 1 A. Photograph of the adaptor with support grid and ring hold- measurement, M3 diaphragm gas meter, V volume flow due to leak - FL ing the filters to be inserted. B Schematic representation of the exper - age, V total volume flow. For the dimensions of the unit see Sup- BGZ imental setup for the validation of the volume streams. v velocity plemental Figure S2 FA at filter inlet, V volume flow, M1 hydrodynamic vane, M2 pressure FA on the tube of any conventional or industrial vacuum cleaner with buffer to collect the material that has fallen off the filter with varying diameters from 2.600 to 3.873 cm. On top of during transportation and handling (details see below). For the adaptor the filter unit is placed. The ring-shaped filter CFU-based analysis of filter-captured microbes, the filter unit carries a grid at the bottom where the filter material is removed from the unit using sterile forceps and directly is placed and then fixed by a hold-down bracket ring. The placed onto suitable growth medium exposing the collection filter unit is closed by a cap (not shown) that is placed on side of the filter to the air. To collect the particles from the top of the filter unit after the sampling has been completed cap, the inner side is rinsed thoroughly with 500 µL sterile to avoid additional microbial contamination of the filter or 1 × phosphate buffered saline (PBS, pH 7.4), 0.01% Tween loss of samples from the filter surface during handling or 20 (Sigma–Aldrich, USA) solution. After gentle shaking for transportation. The whole system can be packed after com- half a minute, this solution is placed directly on top of the plete mounting in a plastic bag and sterilized by gamma filter and distributed over the whole surface of the medium irradiation directly inside the packaging. It thus represents in the petri dish using a Drigalski spatula. This procedure a ready-to-use sterile filter unit that is unpacked at the sam- was found to be optimal not only to collect particles from pling site and placed at the adaptor for each individual aero- the cap but also to evenly distribute microbial cells from sol sampling procedure. Before the first sampling, one filter the filter over the whole area of the medium thus preventing unit is used to calibrate the vacuum cleaner-filter system growth restriction due to exceedingly high colony densities combination for air sampling volume per time interval. For on the filter area. The adaptor and the filter units with the cap this, an anemometer is placed on top of the filter system have been fabricated by low density polyethylene SPB608 mounted already with the adaptor and the vacuum cleaner (Braskem Europe GmbH, Germany) injection molding is started with highest power settings. The settings of the and can be reused after rinsing in 70% ethanol and wash- vacuum cleaner (if available) should be adjusted to yield a ing in a laboratory dish washer. The system has received total sampled air volume between 100 and 150 L per minute intellectual property rights protection and is registered as and sampling times between 1 and 5 min would hence be EP2730912A2. required, depending on the expected bioaerosol load. After this calibration step, the first and all subsequent samplings 2.2 Experimental Setup and Determination can be performed using the same settings. After each indi- of Airstream Correction Factors vidual sampling is completed, the cap is placed and fixed on top of the filter unit which is then put back into the plastic The newly developed system was validated for air stream bag being ready for shipment to the laboratory for analysis. parameters and possible leakage problems that would allow For all sampling operations the same adaptor can be used the air stream passing through the adaptor without passing as only the filter units are exchanged after each round of through the filter. This situation would result in a total air collection. volume measurement by the anemometer that is actually For analysis of the collected airborne material in the labo- higher than the real air volume passing through the filter. ratory the filter unit is demounted in a sterile hood. For this, To know the portion of air passing through the system but the cap is carefully removed from the filter unit and rinsed not through the filter, a leakage analysis was performed and 1 3 Aerosol Science and Engineering (2021) 5:404–418 407 an air volume correction factor was determined. The experi- active filter area. The filter surface is thus composed of an mental setup and the main components for these tests are active and a passive part. Equation (1) calculates the entire described below and graphically shown in Fig. 1B. The gas filter area (A ) but the actual volume flow V through the FA.k velocity at the filter inlet (v ) was measured with a hydro- filter needs to be corrected applying Eq. (2 ). FA dynamic vane (M ) model PCE-TA 30 (PCE Instruments, V = v × A × k . (2) FA.k FA ges Germany). At the drain of the filter holder, a diaphragm gas meter (M ) model BK-G6 (Elster GmbH, Germany) meas- This correction factor k is calculated according to ges ured the total volume flow ( ) including the inlet air by BGZ Eq. (3) and refers to the total volume measured by the ane- leakage before and after the filter holder. To provide a wide mometer but not passing through the filter. range of different volume flows for each filter, a side chan- nel blower model SKV ND-150-3-935 (SKV-tec GmbH, k = k × k . ges Rest FL (3) Germany) was used in vacuum mode. At last, for the deter- The change of condition between in- and outlet of the mination of an alternative measurement method, a pressure sample, can be modelled as isothermal. Therefore, at atmos- measurement device was placed at the pipe connecter (M ). pheric condition, a pressure drop of 40 mbar corresponds to The determination of the volume flow by means of speed a maximum error of 4% (according to ideal gas equation) measurement by a hydrometric vane took place via the equa- of the measured volume flow with the vane anemometer. tion of continuity (1). As mentioned, many other factors (air leakage, grid, turbu- V = v × A, (1) FA FA lences etc.) influence the vane anemometer signal. All fac- tors are summarized by the correction factor k which was ges v denotes the gas velocity measured by hydrometric vane. FA empirically determined (Fig. 2). This factor is only valuable In Fig. 1B this point is marked by the circle M It can be for the used filters and the tested range of volume flow. By assumed that the volume flow, which is determined by correcting the filter surface and the air stream not passing means of the speed of the vane anemometer and the filter through the filter but through the system, the determination surface (A), is higher than the volume flow actually flowing of the real volume flow V is now possible by means of FA.k through the filter: gas velocity measurement. To stabilize the filter during sampling, a grid was inserted Distinct types of membrane filter discs commonly used into the filter carrier. The diameter of the grid and the inner for air-based sampling in occupational hygiene assessment diameter of the ring are the same size (40 mm). However, the (Soo et al. 2016) were used for the physical characterization grid reduces the effective filter area and this fact is consid- of the newly developed filter holders. All filters had a diam- ered by modification of the equation used to determine the eter of 47 mm to fit into the filter holder and included the Fig. 2 Diagram showing the relationship between air stream veloc- holder was according to Fig.  1 and for details on the used filters see ity and the correction factor k with different filter materials, n = 28. descriptions in Materials and Methods ges Experimental setup of establishing the correction factors of the filter 1 3 408 Aerosol Science and Engineering (2021) 5:404–418 following filter types: acrylic copolymer membrane filters in a C-Chip Haemocytometer (Neubauer improved; Incyto, Versapor 1200 (pore size 1.2 µm, Pall Inc. product #66394) South Korea) disposable counting chambers. All spores in and Versapor 3000 (pore size 3  µm, Pall Inc. product the nine big squares were counted using a microscope and #66387), mixed cellulose ester/cellulose nitrate membrane a 400-fold magnification, the mean and final concentration filters GN-4 (pore size 0.8 µm, Pall Inc. product # 64679) were calculated. Homogenous spore suspensions (2.0 × 10 and GN-6 (pore size 0.45 µm, Pall Inc. product # 66191), spores/mL) of T. longibrachiatum in 0.01% Tween 20 were hydrophilic polycarbonate membrane filters Isopore (pore aerosolized and subsequently sampled with the test devices. size 0.4 µm, Merck-Millipore product # HTTP04700), pol- ytetrafluoroethylene (PTFE) membrane filters type TF1000 2.3.2 Cultivation (pore size 1 µm, Pall Inc. product # 66155), type Zefluor 0.45 (pore size 0.45 µm, Pall Inc. product # P5PQ047), and For sampling in the bioaerosol chamber modified HYCON- type Zefluor 2 (pore size 2 µm, Pall Inc. product #P5PJ047). YM medium (RCS-T; peptone 6 g/L; D (+)-glucose mono- hydrate 15  g/L; malt extract 5  g/L; yeast extract 1  g/L; 2.3 Air Sampling agar–agar 16 g/L; pH 7.3 ± 0.2; 0.1% Triton™ X-100) was used. For the field study, microbial particles were cultivated The newly developed filter system was tested under con- on malt extract agar (MEA; Merck, United States); MEA trolled conditions in a bioaerosol test chamber (Pogner et al. containing Rose Bengal (MEA-BR; 25 mg/L, respectively, 2019) and during a field study with different indoor mold 50  mg/L Rose Bengal); dichloran glycerol agar (DG18; situations. In both cases the MAS-100 NT impactor was Merck, United States); and RCS-T medium. No significant used as a reference device for testing the suitability of the differences were detected for the tested Rose Bengal concen- novel filter holders for bioaerosol sampling. trations (data not shown). Colony forming units (CFUs) were counted after 6–7 days of incubation at room temperature 2.3.1 Bioaerosol Chamber (22 °C). CFUs were converted to CFU per m sampled air (CFU/m ). The sampling conditions in the bioaerosols chamber and the according experimental setup used for this study are based 2.3.3 Impaction Sampling on Pogner et al. 2019. In short, the test system that was used for the biological validation of the sampling device, Air sampling with the MAS-100 NT (MAS) (MBV, Swit- comprised of the CCB 3000 bioaerosol chamber (Calibra- zerland) was performed with a constant airflow of 100 L/ tion Chamber for Bioaerosols 3000, Palas, Germany) and min for 2 min for the bioaerosol chamber and field study an LSA nebulizer (Liquid Sparging Aerosolizer, CH Tech- sampling. 18 replicates were conducted for the bioaerosol nologies, United States). The bioaerosol chamber features a chamber experiment. For the field study 4–6 replicates were laminar airflow, even particle distribution and a testing area taken per sampling campaign, site, and medium. A statistical of 0.57  m . The MAS-100 NT and the filter holder of the correction of the impactor was not needed, since CFUs were air filtration-based system were placed in the chamber. The well within the countable range (Ghosh et al. 2015; Mandal casing of the vacuum cleaner was placed outside and was and Brandl 2011). connected to the filter holder through a tube connection in the chamber wall. For the spore suspensions, fungal strains 2.3.4 Filtration‑Based Microbial Sampling were cultivated on malt extract agar (Merck, United States) for 2–3 weeks at room temperature (22 °C). Trichoderma The filter system combined with a vacuum cleaner (VAC) longibrachiatum was used as test organism, since it was used was used for air filtration sampling with different mem- to validate the CCB 3000 bioaerosol chamber as described in brane filter types commonly employed in bioaerosol sam- Pogner et al. 2019 and its conidia can be aerosolized without pling: Versapor 1200 acrylic copolymer membrane disc detergent. As described previously, the use of detergents in filters (V 1200; 47 mm, pore size 1.2 µm, Pall Inc., United aerosolization creates a high load of unspecific particle back - States), cellulose nitrate/mixed cellulose ester filters (GN- ground and thus disqualifies parallel control measurements 6, 47 mm, pore size 0,45 µm, both from Pall Inc., United with the particle counter installed inside the chamber. Har- States), and polycarbonate filters (PC; Isopore membrane, vesting of the spores was performed by taking mycelia and 47 mm, pore size 0.4 µm, Merck-Millipore, Germany). Air spores from the plate with a spatula, adding 0.01% Tween filtration-based sampling for the field study was performed 20 to the plates, vortexing and filtering the suspensions with Versapor 1200 filters (V 1200). A series of different air through sterilized glass wool to remove mycelial cells. The velocities was measured with an impeller anemometer (air- spore suspension’s concentrations (given in spores/mL) were flow meter PCE-TA 30, PCE Instruments, Germany) for all determined by counting a 10 µL aliquot of a proper dilution filters used in this study and according air sampling (L/min) 1 3 Aerosol Science and Engineering (2021) 5:404–418 409 were established (Table ST1). By measuring the air velocity on the media, the sampling device or a combination of these (m/s) of each vacuum cleaner prior to air sampling, the cor- factors. responding sampling volume can be calculated according to Table S1 independent from the performance and type of the 2.3.6 Field Study Fungal Community Spectra vacuum cleaner. For the bioaerosol chamber measurements a “Siemens Super XS dino e, 2200 W” vacuum cleaner (Sie- For the field study, CFUs were counted and classified mens, Germany) was used. Based on the same air velocity according to the groups Alternaria, Cladosporium, Asper- of the vacuum cleaner (0.89 m/s), sampling was performed gillus/Penicillium, Aureobasidium, Fusarium, Neurospora, for 3 min for each of the tested filters (Versapor 1200 fil- Rhizopus/Mucor, Trichoderma, Chaetomium, Epicoccum, ter, Polycarbonate filter, and Cellulose Nitrate filter). Dur - Botrytis, and yeast. The selection of these taxa was based ing the field study sampling was conducted with Versapor on literature indicating frequently occurring outdoor and 1200 filters (V 1200) and a “Simpex 17730, 800 W” (Sim- indoor airborne fungal genera (Bowers et al. 2013; Fierer pex, United States) vacuum cleaner for 3 min 20 s at an air et  al. 2008; Hyvärinen et al. 2001; Lymperopoulou et al. velocity of 0.80 m/s. Five to 6 replicates on 2 independent 2016; Yamamoto et al. 2012). To enable a fast and efficient sampling days were sampled during for the bioaerosol cham- analytical evaluation, the taxa Aspergillus/Penicillium and ber experiments. Two replicates on 4 independent sampling Rhizopus/Mucor were combined in 1 classification due to campaigns were taken per sampling site and medium during their high morphological similarities, as suggested by other the field study. Previously determined correction factors for studies (Li and Kendrick 1995). Unidentified fungal colonies each filter material were considered in the calculations (see were classified in the categories "other sporulating fungi" Sect. 2.2). and sterile mycelia. Rankings were determined correspond- ing to the relative abundance within the sample (1 = low 2.3.5 Field Study Sampling Sites abundance; 2 = medium abundance; 3 = dominant). A taxon was considered as dominant if CFUs for that community The field study was conducted at an industrial unit of a spar - fraction exceeded 75% of the total cell counts. Subsequently, kling wine factory. Further details on the sampling site are low and medium abundances were defined accordingly. Data described in Unterwurzacher et al. (2018). The sampling were illustrated in a heat map showing relative abundances sites covered a wine cellar with obvious mold growth, an within one sample. Technical replicates of each sampling office that is primarily used as storage room without indica- day, sampling location, and cultivation medium were tions of mold infestation and an inner courtyard that sepa- combined. rated the indoor sampling sites and served as outdoor refer- ence. During sampling, a minimum distance of 1 m was 2.4 Statistical Analysis and Linear Regression kept between each sampling device and any other object Models (including other sampling devices, walls, and/or person- nel). Relative humidity and temperature were monitored. Statistical analyses were conducted using R version 3.2.4 (R The observed relative humidity and temperature ranges are Core Team 2015). Pearson’s correlation coefficients between listed in Table ST 2. Four independent sampling campaigns sampling devices and environmental factors (temperature were performed between June 2015 and January 2016. In and relative humidity) were calculated using cor.test (x, y). detail, sampling was conducted at 23 June 2015 (sampling Pearson’s coefficients were used in respect to creating first 1), 5 October 2015 (sampling 2), 23 November 2015 (sam- order linear models and avoiding overfitting of these models pling 3) and 27 January 2016 (sampling 4). for comparison between different sampling devices. Normal Besides the wine cellar with obvious mold growth, an distribution of the residuals was tested by the Shapiro–Wilk indoor sampling site (office) without indications of mold test and p values less than 0.05 were considered significant. infestation was chosen to represent both, a highly contami- For the comparison of different sampling devices, multi- nated site as well as a sampling site with low levels of air- dimensional linear models were developed. These models borne mold particles. Outdoor samples were used as ref- consider besides the dier ff ent sampling methods (CFUs) also erence to determine background levels at the location and the factors temperature and relative humidity. The respective to check for distinct differences between the fungal com- coefficients for each factor were estimated by minimizing the munities indoors and outdoors. The MAS-100 NT impactor sum of squared residuals and the assumption that y-intercept (MAS) was used for validation of the results obtained by the is zero. For estimating the regression coefficients of linear newly designed filter holders. Furthermore, 4 different culti- models, the means of 5 technical repeats were considered. vation media were compared, to investigate if certain fungal For linear least squares regression computation, the R func- communities might be underrepresented, either dependent tion “lm” was used. 1 3 410 Aerosol Science and Engineering (2021) 5:404–418 Differences between mean CFU values of different media the false air entry V . The numerical results are listed in FL or sampling devices were calculated using the R function t Fig. S1 and represent the mean of three measurements, each test (x,y). p values less than 0.01 were considered signic fi ant. of it was determined at four different partial loads. The filters Standard deviations of measurements conducted during GN-6 and Isopore (PC) have a comparatively high correction the field study are shown as boxplot. In the boxplot the cal- factor k due to lower porosity and, therefore, a higher pres- FL culated upper and lower quartiles (75th and 25th percen- sure drop of the filter. Results are shown and summarized tile), the maximum and minimum values (upper and lower in Fig. 2. In summary, it was established that even with a whiskers), and the median (excluding outliers) are indicated. simple vacuum cleaner the adaptor system can be used and If data exceeded 1.5 times, the interquartile range of these correct measurements can be performed as air leakage is measurements were considered as outliers (circles). not critical factor for the novel bioaerosol collection system. 3.2 Sampling Device Comparison Under Controlled Conditions 3 Results A bioaerosol chamber was used to establish stable sampling 3.1 Filter System Characterization conditions for the reference sampler (MAS) and the air fil- tration-based system to be validated (VAC) as described To determine the correction factor k , the parameters k ges Rest before (Pogner et al. 2019). Three different filter materials and k were measured individually. For k , deter mination, FL Rest were tested with the air filtration-based sampler in this com- the filter attachment site with the ring fixing the filter and parative study under controlled conditions. Similar sampling the adaptor were glued airtight, so that it can be assumed ̇ concentrations between the distinct filter materials were that the air volume flow due to leakage V is equal to zero. FL detected (162–206 CFU/m ), with the exception of the com- For this experimental setup, k can be calculated using Eq. Rest ̇ parison of the Cellulose Nitrate and the Polycarbonate filter, (4), where v is the gas velocity at the filter inlet, V is FA BGZ where mean CFU/m values were in a comparable range, but the total volume flow, v denotes the velocity measured by FA differences were significant (p value < 0.01). Independently hydrometric vane and A is the filter surface area. from the tested l fi ter materials, the air l fi tration-based system showed highly significant impairment in sampling Tricho- BGZ k = . (4) Rest v × A derma longibrachiatum spores compared to the MAS-100 FA NT impactor (MAS), with on average 66.9% lower CFU The air passing through the system but not through the counts detected by the air filtration system (Fig.  3). Con- filter is termed “false air”. In our setup this results from the sidering the broad stable range of different air velocities difference between the volume flow of the diaphragm gas (roughly 0.7–2.0 m/s) tested during the establishment of meter V and the corrected volume flow of the hydromet- BGZ the filters’ correction factors (Fig. S1) and the comparable ric vane V according to Eqs. (5) and (6). FA.k sampling efficiency with the 2 other tested filter materials in the bioaerosol chamber, in further consequence the Versapor ̇ ̇ ̇ V − V = V , (5) BGZ FA.k FL 1200 filter was used for the field study. and, 3.3 Comparative Field Study FL = false air % . (6) BGZ For further validation of the air filtration-based sampler, a comparative e fi ld study was conducted at the production site The test series for determining the correction factor k , Rest of a sparkling wine factory during 4 independent sampling was carried out with airtight glued filter components. As campaigns. a result, the entry of false air V due to leaks in the filter FL attachment could be excluded and the volume flow of the diaphragm gas meter V can be considered as the actual 3.3.1 Detected Colony Forming Units BGZ o fl w rate through the l fi ter. To determine the false air portion of the filter system, the experiments for the determination of Independent from the sampling site and sampling device, k were repeated with non-bonded components. Identical there was a clear seasonal shift in airborne fungal particles Rest to the test series of the bonded components, the volume flow (Fig. 4). Overall highest CFU values (up to 2054 CFU/m 3 3 of each filter was plotted over the velocities at four different outdoors; 3196 CFU/m wine cellar; 724 CFU/m office) partial loads and a linear regression model was formed. The were measured at sampling campaigns 1 and 2. difference in the volume flows at the same speeds results in 1 3 Aerosol Science and Engineering (2021) 5:404–418 411 While cultivation medium had little influence on CFU counts from filtration-based sampling, signic fi ant die ff rences (p value < 0.01) were found for 41.7% of measurements from the MAS-100 NT (MAS) (Table ST3). Comparing differences in sampling devices, particularly outdoors the MAS-100 NT impactor (MAS) and the air fil- tration-based sampler detected fungal CFUs in a highly simi- lar range (MAS: 2054–230 CFU/m ; VAC: 1618–241 CFU/ m ). In the wine cellar, the air filtration-based sampler measured significantly higher CFU values compared to the MAS-100 NT impactor (MAS: 1731–463 CFU/m ; VAC: 3196 ± 365 CFU/m ). The CFU levels detected in the office were similar between the two sampling systems and ranged between MAS: 703 ± 48 CFU/m and VAC: 724 ± 98 CFU/ m . 3.3.2 Measurement Stability Based on Relative Standard Deviations Fig. 3 Results of the bioaerosol chamber experiments. Compari- Overall, sampling with the MAS-100 NT (MAS) showed son of CFU detection of Trichoderma longibrachiatum spores under a high reproducibility with slightly lower relative standard controlled conditions in a bioaerosol chamber using the filter-adaptor deviations (rsd) of the measurement standard deviations system. The MAS-100 NT (MAS) was used as a reference device compared to the air filtration-based sampler (median of rsd: (n = 18). Different membrane filters were tested for the air filtration- based sampling using the vacuum cleaner-adaptor system (VAC): MAS: 12.8%; VAC: 19.4%) (Fig. 5). Particularly outdoor VAC Versapor: Versapor 1200 filter (n = 5), VAC Cell.Nitr.: cellulose measurements conducted with the air filtration-based sam- nitrate GN-6 filter (n = 6), and VAC Polycarb: Isopore polycarbonate pler varied strongly, while MAS-100 NT data were more filter (n = 6). Asterisk indicates significant difference to MAS refer - robust (median of rsd: MAS: 12.1%; VAC: 24.1%). Highest ence sampler similarities in measurement reproducibility were found in the office with little expected spore counts in the aerosols, where both sampling devices showed relatively high data fluctuations (median of rsd: MAS: 18.4%; VAC: 22.4%). Fig. 4 Results of the field studies. CFU counts detected at a comparative field sampling study at four different sampling days (1–4) conducted with the MAS-100 NT (MAS) and air filtration (VAC; on Versapor 1200 filters) for the tested cultivation media DG18, MEA, MEA-BR, and RCS-T. Results are given for each sampling site. Asterisk indicates significant difference to MAS reference sampler 1 3 412 Aerosol Science and Engineering (2021) 5:404–418 Fig. 5 Measurement reproducibility of the air filtration system. Rela- Fig. 6 Correlations of detected CFU counts between the reference tive standard deviations [rsd (%)] of field samplings were calculated sampler MAS-100 NT (MAS) and the air filtration-based sampler from the technical replicates to the means of the sampling campaign (VAC; on Versapor 1200 filters). All sampling campaigns and culti- for the MAS-100 NT (MAS) and the air filtration-based sampler vation media are shown. Data are indicated dependent on the sam- (VAC; on Versapor 1200 filters). Boxplots include data from four pling locations of the comparative field study (outdoor reference; independent sampling campaigns and four different media, resulting office; wine cellar) and the continuous line represents the overall lin- in 16 data points for each sampling location and sampler. In the box- ear correlation plot the calculated upper and lower quartiles (75th and 25th percen- tile), the maximum and minimum values (upper and lower whiskers), the CFU counts from another device in this study, consid- and the median (excluding outliers) are indicated. If a given SD value exceeded 1.5 times the interquartile range, this value was considered ering environmental temperature and relative humidity: as outlier (circle). Values used for statistical analyses shown in the CFU counts(sampling device 1) boxplots are listed in Supplementary Table ST 4 = A × CFU counts(sampling device 2) CFU Contrary to that, both samplers displayed the lowest rela- + B × Temperature C temp. tive standard deviations in the wine cellar, which generally + C × relative humidity [%]. rel.hum. (7) exhibited high CFU levels due to the obvious indoor mold infestation (median of rsd: MAS: 9.3%; VAC: 14.6%). Besides CFU counts, only the influence of humidity 3.3.3 Sampler Correlations and Linear Regression Models was statistically significant. Temperature was of minor importance. The herein presented correlations are valid for A linear regression model for comparison of sampling this study. Further comparative measurement campaigns devices and methodologies based solely on CFU counts would be needed for more general correlations. Also, poly- showed already high comparability between the sampling nomial functions were investigated but could not bring a devices (MAS-100 NT and air filtration) with an R value significant improvement to our models (data not shown). of 0.90 (Fig. 6). Furthermore, Pearson’s correlations (over- Measurements taken outdoors could not sensibly be fitted all data Shapiro–Wilk test of normality, p value > 0.05) at each sampling site substantiated these strong correlations Table 1 Regression coefficients for linear models (Eq.  7) to compare between the compared air samplers (outdoors: cor = 0.96, sampling devices based on measured values in this comparative study p value < 0.01; cellar: cor = 0.89, p value < 0.01; and office: Sampling Regression coefficients Adj R p Shapiro– cor = 0.77, p value < 0.01). device Wilk Furthermore, we developed multi-dimensional linear A B C CFU Temp Humid models that implement also the factors of temperature VAC → MAS 1.026*** − 2.638 5.376*** 0.9473 0.939 and relative humidity (Supplementary Table ST2). Con- sidering these additional parameters, the adjusted R value Significance codes: ***0.001; **0.01; *0.05 reached nearly 0.95 (Table 1). Equation 7 allows calculat- a Shapiro–Wilk test of normality, p values > 0.05 indicating normality ing CFU counts from a specified device in our study given are highlighted in bold 1 3 Aerosol Science and Engineering (2021) 5:404–418 413 into these models as there may be influencing factors that medium. While outdoors especially Cladosporium and were not measured in our experiments (e.g., wind velocity Alternaria species were detected, in the wine cellar Aspergil- and air turbulence). lus/Penicillium species were dominant, indicating an indoor mold infestation. In the vacant office, besides typical outdoor 3.3.4 Culture‑Based Fungal Community Spectra taxa Cladosporium and Alternaria also Aspergillus/Penicil- lium species were detected more frequently, which could be Besides CFU values, the influence of cultivation media and explained by whirled up dust during sampling rather than by sampling devices on the detected fungal community spectra indoor mold growth. was investigated during the field study. To cover all possible Generally, fungal community compositions sampled with underrepresented fungal genera, 4 different sampling media the MAS-100 NT (MAS) impactor showed slightly higher (DG18; MEA; MEA-BR; RCS-T) with distinct cultivation diversity at all sampling locations. For example, colonies characteristics were chosen. Community composition was belonging to the fungal taxa Aureobasidium, Trichoderma, highly influenced by both, sampling method and growth and Epicoccum were almost exclusively detected with the Fig. 7 Community spectra of cultivated airborne microbial particles The heat map displays relative abundances of the fungal communities sampled with the MAS-100 NT (MAS) and the air filtration-based detected within the sample. The color code is indicated. Yellow: high- system (VAC) during the field study. All sampling days (1–4), sam- est abundance; dark blue: lowest abundance; black: below detection pling locations (outdoor reference; office; wine cellar) and different limit (bdl). Mean values of replicates are represented (color figure cultivation media (DG18, MEA, MEA-BR; RCS-T) are illustrated. online) 1 3 414 Aerosol Science and Engineering (2021) 5:404–418 MAS-100 NT (MAS). For the most dominant genera such NT control device as defined in our previous study (Pogner as Alternaria, Cladosporium, and Aspergillus/Penicillium et al., 2019). As outlined in the Sect.  2 Trichoderma was similar abundances were found with both sampling devices used because it is one of the few species whose spores can (Fig. 7). be aerosolized without detergent and thus allows parallel control measurements with a particle counter installed inside the chamber. Moreover, in the subsequent field sampling 4 Discussion comparative studies were broad in terms of conditions, loca- tions, and expectable fungal biodiversity. Here, we present the development and evaluation of a new Generally, the testing in the bioaerosol chamber demon- filter-based bioaerosol sampling system that can be operated strated measurement stabilities of viable T. longibrachia- with commercially available vacuum cleaners as air pumps. tum spores similar to values reached by the MAS-100 NT, It opens the possibility to perform multiple sampling cam- suggesting high detection reproducibility of the novel air paigns in parallel in a short period of time at many die ff rent, filtration system under conditions without wind effects. In geographically distant locations. Such samplings are done future, more microbial species will be compared between for instance for epidemiologic studies to screen pathogens the filter-based sampler and a reference sampler in the bio- or monitor antibiotic resistance genes by molecular biol- aerosol chamber to gain better insights into possible bias for ogy analysis. Until now, large-scale comparative samplings certain types of microbes. are not routinely performed because at each of these distant As reported by Elmashae and coworkers (Elmashae et al. sampling locations, a standard bioaerosol sampler is needed 2017), which tested different filter materials accordingly to and the associated costs usually exceed the budget available identify airborne bacterial cells, our results showed similar for such studies. The adaptor and sterile filter unit composite detection rates with all tested filter materials. It has to be serve as a feasible option for routine analytics as the system mentioned, that pore sizes of the different membrane fil- is highly cost-effective compared to other sampling devices, ter discs tested in the bioaerosol chamber were not identi- since beside a vacuum cleaner that is usually available and cal, but a similar size range (Versapor 1.2 µm pore size; an anemometer, no further equipment is required. The phys- Cellulose Nitrate and Polycarbonate 0.8  µm pore sizes). ical tests of the sampling system with different air filters Nevertheless, considering that typical fungal spores feature resulted in the formulation of a correction factor for each sizes from approx. 1–30 µm (Yoo et al. 2017), the effect of filter to be applied. This factor in combination with a simple the different pore sizes is expected to be rather small. This anemometer gave consistent sampling volumes in each of the would be in agreement with the findings of Soo et al. ( 2016), tests run. Therefore, the combination of the filter-adaptor who detected similar particle collection efficiencies in the and vacuum cleaner can thus be regarded as reliable bio- range of 1 µm pore sizes and strong deviations only com- aerosol sampling system. An uncertainty of the system that pared to higher pore sizes among different commonly used has so not been tested in the long run may be a reduction of air filter materials. vacuum generation by the vacuum cleaner. The performance The new filter system was also functional in displaying of the system might suffer from reduced electric engine per - expected seasonal fluctuations in cultivatable fungal cells. formance after a long sampling campaign. However, field It is well known that the composition and biodiversity of data from more than 300 individual test runs as presented airborne microbial cells as well as their concentrations here gave quite consistent CFU counts in comparison to the change strongly with seasons (Bowers et al. 2013; Frankel reference sampler and thus we do not foresee large problems et al. 2012a; Heo et al. 2014; Matthias-Maser et al. 2000). due to reduction of performance of the vacuum cleaner used As reported before by Bowers and colleagues (Bowers et al. for sampling. 2013), also our results showed highest fungal quantities Although the biological sampling efficiency was com- between summer and fall and lowest in winter. The diverse parable to the reference device in our field studies, under sampling time points covering different seasons were cho- controlled conditions in a bioaerosol chamber, the air filtra- sen for this study to determine if not only number but also tion-based sampler showed significantly lower CFU-based biodiversity of monitored microbes correlate between our collection efficiency of aerosolized Trichoderma longibra- device and the reference sampler. Strong overall correlations chiatum spores. The susceptibility of the sampled micro- between the tested and the reference devices indicate high organism due to diverging sampling stress pursued by the measurement reliability of the new filter holder system for die ff rent sampling principles might explain this discrepancy environmental samples (Ghosh et al. 2015; Kalogerakis et al. (Wang et al. 1999). Although a variety of different species 2005; Yao and Mainelis 2006). could have been tested with the new filter system in the To investigate if certain fungal species are underrepre- chamber, we decided to perform this first step in validation sented in the newly developed sampling system, four dif- only under the most suitable conditions for our MAS-100 ferent cultivation media were compared during the field 1 3 Aerosol Science and Engineering (2021) 5:404–418 415 study. The tested cultivation media covered highly divergent filtration system. Higher susceptibility to desiccation stress growth properties including different carbon and nitrogen during air filtration might explain the underrepresentation sources, water activities and inhibitory compounds. The air of more desiccation-sensitive species (Ghosh et al. 2015; filters detected comparable CFU counts on all tested culti- Wang et al. 2001). vation media, which was not the case for the MAS-100 NT. Overall, the field study indicated a strong and highly sig- A reduction in biodiversity due to higher sampling stress nificant correlation between the newly designed filter holders (Zhen et al. 2018) during filtration (see below) could explain and the well-established impactor system. The implemen- the minor influence of cultivation media on enumeration of tation of multi-dimensional linear models for the indoor fungal propagules. Fungi resistant to the stress imposed by measurements considered the environmental factors tem- filtration probably grow equally well on different growth perature and relative humidity, which further strengthened media. the correlation. Given the results from these models, it can There was also no significant bias towards indoor or be deduced that especially relative humidity has a significant outdoor locations. Particle counts detected outdoors and in impact on the comparability between the different sampling the office were in a highly similar range for both sampling methods used in this study. The temperature did not show a devices. In the wine cellar, particularly at sampling cam- significant influence on the measurement correlations. Both paigns 1 and 2, the filter-based system reached higher CFU bioaerosol samplers were subjected to some species related counts compared to the MAS-100 NT. Since the community restrictions in detecting viable fungal cells, but overall the spectra of the office and the outdoor reference are more alike CFU-based results were comparable and the CFU regression as shown in another study conducted at this site (Unterwur- coefficient of 1.026 (p value < 0.01) indicates the reproduc- zacher et al. 2018), discrepancies in particle detections in ibility of the compared sampling devices during environ- the wine cellar might be again a result of species related mental conditions. underrepresentation. Obviously, presented results are valid for the sampling However, one weakness of the newly developed system settings chosen in this study, different sampling durations might be the higher variability of detected fungal counts. or airflow rates might lead to diverging correlations, since Overall, relative standard deviations were higher for the air sampling settings are crucial for the overall performance of filters compared to the impactor at the chosen conditions bioaerosol samplers (Grinshpun et al. 2007; Han et al. 2015; and experimental design, i.e., two technical replicates per Uhrbrand et al. 2017). Although a broad range of environ- cultivation media, per sampling site, and per sampling date. mental conditions, including different seasons, indoor sam- Although this sampling strategy still concurs with official pling sites with high, respectively, low fungal particle levels directives and guidelines, e.g., DIN ISO 16000-16 (2008) and outdoor reference measurements were covered within the comparatively low number of repeats might explain this experimental setup, further studies at different sampling higher relative standard deviations. Furthermore, filters need sites are needed to verify these finding. One limitation of additional manipulation steps after sampling, while plates the herein presented field study is the rather small number from the MAS-100 NT impactor are directly incubated after of technical replicates, which possibly explains the higher sampling. Handling of filters can potentially increase varia- relative standard deviations compared to the MAS-100 NT tion in enumeration of fungal propagules. reference device. Similar to other comparative studies of bioaerosol sam- plers, we also found differences in community composition depending on the sampling device (Lukaszuk et al. 2017; 5 Conclusion Unterwurzacher 2016). Nevertheless, it cannot be excluded that particle dispersal not only occurred through the overall The findings underline the applicability of the novel vacuum- sampling activity (Meadow et al. 2014) but especially dur- cleaner-based filter sampling system to detect viable fungal ing the use of the vacuum cleaner (Corsi et al. 2008; Knibbs cells and spores in bioaerosols. The system is largely equiva- et al. 2012). lent to other commonly used samplers as it gave similar results Sampling with the MAS-100 NT impactor showed as the reference MAS-100 NT impaction sampler. The advan- slightly higher diversity at all investigated sites. Although tage of this system is that (1) no extra air pump or collector most dominant taxa including Alternaria, Cladosporium, needs to be purchased as vacuum cleaners are usually avail- and Aspergillus/Penicillium were detected in a similar range, able, (2) materials used for collection (sterile filters) endure other taxa such as Aureobasidium, Trichoderma, and Epico- harsh temperature conditions during storage and thus have ccum were almost exclusively detected with the MAS-100 a long shelf life, and (3) the simplicity of the system allows NT impactor. This is consistent with the findings in the bio- large number sampling campaigns to be run in parallel at very aerosol chamber experiment, where Trichoderma longibra- distant locations (e.g., broad antibiotic resistance sampling chiatum spores were strongly underrepresented by the air campaigns). A weakness of the system may be the need of 1 3 416 Aerosol Science and Engineering (2021) 5:404–418 as you give appropriate credit to the original author(s) and the source, an anemometer to calibrate the air volume sampled per time provide a link to the Creative Commons licence, and indicate if changes unit, a possible underrepresentation of some genera depend- were made. The images or other third party material in this article are ing on the chosen filters and a slightly higher variability of included in the article’s Creative Commons licence, unless indicated the obtained results due to changing power capacities of the otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not vacuum cleaners. However, these slight disadvantages may be permitted by statutory regulation or exceeds the permitted use, you will compensated by opening the possibility to perform large paral- need to obtain permission directly from the copyright holder. To view a lel sampling campaigns at distant locations. Such scientifically copy of this licence, visit http://cr eativ ecommons. or g/licen ses/ b y/4.0/ . valuable endeavors would not be economically feasible if at each of these locations a costly commercial sampler needs to be purchased. Other bioaerosol compounds such as spore- References forming bacteria or pollen can equally be sampled with the described system once validation studies with these compo- Aizenberg V, Reponen T, Grinshpun SA, Willeke K (2000) Perfor- mance of Air-O-Cell, Burkard, and Button Samplers for total nents have been performed similar to the work presented here. enumeration of airborne spores. Am Ind Hyg Assoc J 61(6):855– 864. https:// doi. or g/ 10. 1202/ 0002- 8894(2000) 061% 3c0855: Supplementary Information The online version contains supplemen- POAOCB% 3e2.0. CO;2 tary material available at https://doi. or g/10. 1007/ s41810- 021- 00110-9 . An HR, Mainelis G, Yao M (2004) Evaluation of a high-volume port- able bioaerosol sampler in laboratory and field environments. Acknowledgements The authors wish to express their gratitude to Indoor Air 14(6):385–393. https:// doi. org/ 10. 1111/j. 1600- 0668. Dragana Bandian from Austrian Institute of Technology GmbH (AIT) 2004. 00257.x and the AQA team for technical support and sampling assistance. This Bernstein JA, Alexis N, Bacchus H, Bernstein IL, Fritz P, Horner E, work was supported by the Lower Austria Corporation for Research Li N, Mason S, Nel A, Oullette J, Reijula K, Reponen T, Seltzer and Education (NFB; Grant LS12-011) and by the DGUV together with J, Smith A, Tarlo SM (2008) The health effects of nonindustrial the AUVA (Grant FF-FP-0337). Special thanks to the occupants of the indoor air pollution. J Allergy Clin Immunol 121(3):585–591. site, where sampling was conducted. https:// doi. org/ 10. 1016/j. jaci. 2007. 10. 045 Bowers RM, Clements N, Emerson JB, Wiedinmyer C, Hannigan Author contributions Study design: SSG, MG, SB, CP, GT, and JS. MP, Fierer N (2013) Seasonal variability in bacterial and fungal Performed research: VU, MB, CP, AK, and HB. Analysis of data: SB, diversity of the near-surface atmosphere. Environ Sci Technol GT, HB, SSG, CP, JS, and MG. Writing of manuscript: VU, SSG, CP, 47(21):12097–12106. https:// doi. org/ 10. 1021/ es402 970s GT, HB, JS, and MG. All authors read and approved the manuscript. Burton NC, Grinshpun SA, Reponen T (2007) Physical collection effi - ciency of lt fi er materials for bacteria and viruses. Ann Occup Hyg 51(2):143–151. https:// doi. org/ 10. 1093/ annhyg/ mel073 Funding Open access funding provided by University of Natural Chang CW, Hwang YH, Grinshpun SA, Macher JM, Willeke K (1994) Resources and Life Sciences Vienna (BOKU). This work was sup- Evaluation of counting error due to colony masking in bioaerosol ported by the Lower Austria Corporation for Research and Education sampling. Appl Environ Microbiol 60:3732–3738 (NFB; Grant LS12-011) and by the DGUV together with the AUVA Corsi RL, Siegel JA, Chiang C (2008) Particle resuspension during the (Grant FF-FP-0337). use of vacuum cleaners on residential carpet. J Occup Environ Hyg 5(4):232–238. https://doi. or g/10. 1080/ 15459 62080 19011 65 Code availability Not applicable. Dannemiller KC, Gent JF, Leaderer BP, Peccia J (2016) Influence of housing characteristics on bacterial and fungal communities in Declarations homes of asthmatic children. Indoor Air 26(2):179–192. https:// doi. org/ 10. 1111/ ina. 12205 Deacon LJ, Pankhurst LJ, Drew GH, Hayes ET, Jackson S, Longhurst Conflict of interest The authors declare a potential conflict of interest PJ, Longhurst JWS, Liu J, Pollard SJT, Tyrrel SF (2009) Par- as a member of the company that may in future bring the sampling ticle size distribution of airborne Aspergillus fumigatus spores system to market is a coauthor of this manuscript. All authors declare emitted from compost using membrane filtration. Atmos Environ that this fact, however, has not influenced the design or interpretation 43:5698–5701. https:// doi. org/ 10. 1016/j. atmos env. 2009. 07. 042 of the results and that the research was conducted in the absence of any DIN-ISO16000-16 (2008) Indoor air. Part 16: detection and enumera- commercial or financial interest. tion of moulds—sampling by filtration, vol 16000-16. Interna- tional Standards Organization Ethics approval Not applicable. DIN-ISO13205-1:2014 (2014) Workplace atmospheres-Assessment of performance of instruments for measurement of airborne particle Consent to participate Not applicable. concentrations German Version. International Standards Dybwad M, Skogan G, Blatny JM (2014) Comparative testing and Consent for publication Not applicable. evaluation of nine different air samplers: end-to-end sampling efficiencies as specific performance measurements for bioaerosol Research involving human and animal participants This article does applications. 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Development and Validation of a Simple Bioaerosol Collection Filter System Using a Conventional Vacuum Cleaner for Sampling

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Springer Journals
Copyright
Copyright © The Author(s) 2021
ISSN
2510-375X
eISSN
2510-3768
DOI
10.1007/s41810-021-00110-9
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Abstract

Although numerous bioaerosol samplers for counting and identifying airborne microorganisms are available, the consid- erably high purchase and maintenance costs for the sampler often prevent broad monitoring campaigns for occupational or environmental surveillance of bioaerosols. We present here a newly developed simple adapter and filter system (TOP filter system) designed to collect bioaerosol particles from a defined air volume using conventional vacuum cleaners as air pumps. We characterized the physical properties of the system using air flow measurements and validated the biological performance. The culture-based detection capacities for airborne fungal species were compared to a standard impaction sampler (MAS-100 NT) under controlled conditions in a bioaerosol chamber (using Trichoderma spores as the test organ- ism) as well as in the field. In the chamber, an overall equivalent detection capacity between all tested filters was recorded, although a significant underrepresentation of the TOP filter system for Trichoderma spores were seen in comparison to the MAS-100 NT. In a comparative field study (n = 345), the system showed similar biological sampling efficiencies compared to the MAS-100 NT impactor, only the diversity of identified fungal communities was slightly lower on the filters. Thus, the system is suitable for large-scale environmental sampling operations where many samples have to be taken in parallel at a given time at distant locations. This system would allow endeavors such as antibiotics resistance monitoring or hygiene surveys in agricultural or occupational settings. Keywords Bioaerosol sampling · Air filtration · Sampling device validation · Field study · Bioaerosol chamber · Indoor mold assessment Abbreviations MAS MAS-100 NT (Microbial Air Sampler) bdl Below detection limit MCE Mixed cellulose esters CFU Colony forming unit MEA Malt extract agar MCE Mixed cellulose ester MEA-BR Malt extract agar containing Rose Bengal DG18 Dichloran glycerol agarPTFE Polytetrafluoroethylene (TF) RCS-T Modified HYCON-YM medium sd Standard deviation * Sabine Strauss-Goller VAC Air filtration by filter holder and vacuum sabine.goller@boku.ac.at cleaner Fungal Genetics and Genomics Laboratory, Department of Applied Genetics and Cell Biology, Institute of Microbial Genetics, BOKU-University of Natural Resources and Life 1 Introduction Sciences, Tulln, Austria Center for Health and Bioresources, AIT-Austrian Institute Modern life in industrialized countries dictate that people of Technology GmbH, Tulln, Austria usually spend more than 80% of their daily time indoors Department of Material Sciences and Process Engineering, (Bernstein et al. 2008; Klepeis et al. 2001; Normand et al. Institute of Chemical and Energy Engineering, 2015). Hence, healthy indoor air and monitoring indoor air BOKU-University of Natural Resources and Life Sciences, quality is of great importance and public concern. Exposure Vienna, Austria to microbial air pollutants present as bioaerosols has been AQA Umweltanalytik GmbH, Klosterneuburg, Austria Vol:.(1234567890) 1 3 Aerosol Science and Engineering (2021) 5:404–418 405 related to a wide range of diseases such as allergy, asthma types include membrane filters composed of gelatin (Wu or respiratory disorder (Dannemiller et al. 2016; Madsen et al. 2010), polymer layers and fibrous filters consisting of et al. 2016; Nevalainen et al. 2015; Osborne et al. 2015). cellulose, glass or variant copolymers (Burton et al. 2007; Increased awareness of such potential adverse health effects Haig et al. 2016; Miaskiewicz-Peska and Lebkowska 2012; caused by airborne microorganisms raised the interest in Raynor et al. 2011). Compared to impactors, filtration-based reliable methods for the characterization and collection of samplers cover a wider range of particle sizes with high col- bioaerosols. In addition, fungi and bacteria are prominent lection efficiency depending on the pore size of the mem- pathogens and monitoring of these microbes in aerosols and brane (Aizenberg et al. 2000). One advantage of air filtra- testing their antibiotic or antimycotic resistance profiles is tion is the compatibility with various analytical techniques an important task in public health management (Jones and including cultivation, DNA-based, biochemical, immuno- Brosseau 2015). chemical, and gravimetrical analyses. Generally, high vol- Besides air filtration-based sampling, where airborne ume sampling is desired but particularly for a culture-based particles are drawn through a filter holder and captured on analysis, long-term air filtration may also lead to overloading filters, impaction and impingement are among the most fre- and extended sampling stress (Eduard and Heederik 1998; quently used and well-established air sampling techniques Wang et al. 2001). (Ghosh et al. 2015). Mostly, microbial analysis of bioaero- Besides validation of the physical collection efficiency sol loads relies on cultivation-based techniques that provide according to directives such as DIN ISO 13205-1 (2014), an estimation of culturable microorganisms expressed as air samplers are often compared in field studies to charac- “colony forming units” (CFUs) (Chang et al. 1994; Ghosh terize their overall performance (An et al. 2004; Frankel et al. 2015). Although culturable and non-culturable micro- et al. 2012b; Wang et al. 2015). Discrepancies between such organisms are usually present in bioaerosols international comparative measurements occur due to the generally low standards for the detection and enumeration of these organ- microbial particle concentrations in the environment, parti- isms usually rely on CFU counting and species determina- cle agglomerations, and the unequal distribution of bioaero- tion based on morphological traits. If using a culture-based sols in the air (Fierer et al. 2008; Ghosh et al. 2015; Nazaroff analysis, it has to be considered that an accurate enumeration 2004, 2016). Specific technical limitations of each sampling of the collected airborne microorganisms strongly depends device might amplify these differences. To compensate for on parameters such as cultivability and viability of the such uncertainties, various bioaerosol chambers have been microbial species and the selection of an appropriate culti- implemented to enable stabilized sampling condition (Dyb- vation medium and growth conditions, sampling method and wad et al. 2014; Estill et al. 2008; Feather and Chen 2003; analytical strategy. Furthermore, environmental factors such Kesavan and Sagripanti 2015; Pogner et al. 2019; Simon and as relative humidity, UV radiation, and temperature show a Duquenne 2013; Wang et al. 2001). significant impact on the survival rate of the microorganisms In the present study, we validated a novel filter-based and, therefore, influence a cultivation-based overall assess- sampling system that uses conventional vacuum cleaners as ment (Juozaitis et al. 1994; Tseng and Li 2005; Zhen et al. air pumps. The initiative to develop such a system comes 2018). For air filtration-based sampling, desiccation effects from the necessity of parallel air samplings in large sam- are likely to impair the survival of collected microorgan- ple numbers at many different locations in a short period of isms and might lead to an underestimation of the microbial time. Typically, such sampling campaigns are performed in particles present, particularly of the sensitive ones, such as hygiene surveys or when antibiotic resistance profiles need vegetative cells. (Ghosh et al. 2015; Haig et al. 2016; Wang to be determined over a wide geographic area at a given et al. 2001). Spore forming bacteria and fungi, which are time point. For this, many samplers need to be employed at highly resistant to environmental stresses, are more ame- each of the distant locations and it is financially not feasible nable to monitoring with filtration sampling. Only recently, to equip each of the operators with one of the costly com- molecular methods such as DNA-based quantification mercial bioaerosol samplers. The system described here is of microbes or quantitative biochemical assays (Kespohl low-cost and easy to handle and would thus be well suited et al. 2013; Zahradnik and Raulf 2019) and fungal-specific for such large sampling campaigns. enzyme assays (Reeslev et al. 2003) are starting to be com- monly used for routine measurements and will be imple- mented in ISO Standards worldwide (Rylander et al. 2010; 2 Materials and Methods Unterwurzacher et al. 2018). Air filtration is usually easy to accomplish and depend- 2.1 Description of the Adaptor and Filter System ing on the reason for sampling, a broad spectrum of filter materials featuring different characteristics and pore sizes The system consists of a cylindrical adaptor with a conical are available (Deacon et al. 2009). Commonly used filter inner wall (see Fig. 1A) that is designed to place the adaptor 1 3 406 Aerosol Science and Engineering (2021) 5:404–418 Fig. 1 A. Photograph of the adaptor with support grid and ring hold- measurement, M3 diaphragm gas meter, V volume flow due to leak - FL ing the filters to be inserted. B Schematic representation of the exper - age, V total volume flow. For the dimensions of the unit see Sup- BGZ imental setup for the validation of the volume streams. v velocity plemental Figure S2 FA at filter inlet, V volume flow, M1 hydrodynamic vane, M2 pressure FA on the tube of any conventional or industrial vacuum cleaner with buffer to collect the material that has fallen off the filter with varying diameters from 2.600 to 3.873 cm. On top of during transportation and handling (details see below). For the adaptor the filter unit is placed. The ring-shaped filter CFU-based analysis of filter-captured microbes, the filter unit carries a grid at the bottom where the filter material is removed from the unit using sterile forceps and directly is placed and then fixed by a hold-down bracket ring. The placed onto suitable growth medium exposing the collection filter unit is closed by a cap (not shown) that is placed on side of the filter to the air. To collect the particles from the top of the filter unit after the sampling has been completed cap, the inner side is rinsed thoroughly with 500 µL sterile to avoid additional microbial contamination of the filter or 1 × phosphate buffered saline (PBS, pH 7.4), 0.01% Tween loss of samples from the filter surface during handling or 20 (Sigma–Aldrich, USA) solution. After gentle shaking for transportation. The whole system can be packed after com- half a minute, this solution is placed directly on top of the plete mounting in a plastic bag and sterilized by gamma filter and distributed over the whole surface of the medium irradiation directly inside the packaging. It thus represents in the petri dish using a Drigalski spatula. This procedure a ready-to-use sterile filter unit that is unpacked at the sam- was found to be optimal not only to collect particles from pling site and placed at the adaptor for each individual aero- the cap but also to evenly distribute microbial cells from sol sampling procedure. Before the first sampling, one filter the filter over the whole area of the medium thus preventing unit is used to calibrate the vacuum cleaner-filter system growth restriction due to exceedingly high colony densities combination for air sampling volume per time interval. For on the filter area. The adaptor and the filter units with the cap this, an anemometer is placed on top of the filter system have been fabricated by low density polyethylene SPB608 mounted already with the adaptor and the vacuum cleaner (Braskem Europe GmbH, Germany) injection molding is started with highest power settings. The settings of the and can be reused after rinsing in 70% ethanol and wash- vacuum cleaner (if available) should be adjusted to yield a ing in a laboratory dish washer. The system has received total sampled air volume between 100 and 150 L per minute intellectual property rights protection and is registered as and sampling times between 1 and 5 min would hence be EP2730912A2. required, depending on the expected bioaerosol load. After this calibration step, the first and all subsequent samplings 2.2 Experimental Setup and Determination can be performed using the same settings. After each indi- of Airstream Correction Factors vidual sampling is completed, the cap is placed and fixed on top of the filter unit which is then put back into the plastic The newly developed system was validated for air stream bag being ready for shipment to the laboratory for analysis. parameters and possible leakage problems that would allow For all sampling operations the same adaptor can be used the air stream passing through the adaptor without passing as only the filter units are exchanged after each round of through the filter. This situation would result in a total air collection. volume measurement by the anemometer that is actually For analysis of the collected airborne material in the labo- higher than the real air volume passing through the filter. ratory the filter unit is demounted in a sterile hood. For this, To know the portion of air passing through the system but the cap is carefully removed from the filter unit and rinsed not through the filter, a leakage analysis was performed and 1 3 Aerosol Science and Engineering (2021) 5:404–418 407 an air volume correction factor was determined. The experi- active filter area. The filter surface is thus composed of an mental setup and the main components for these tests are active and a passive part. Equation (1) calculates the entire described below and graphically shown in Fig. 1B. The gas filter area (A ) but the actual volume flow V through the FA.k velocity at the filter inlet (v ) was measured with a hydro- filter needs to be corrected applying Eq. (2 ). FA dynamic vane (M ) model PCE-TA 30 (PCE Instruments, V = v × A × k . (2) FA.k FA ges Germany). At the drain of the filter holder, a diaphragm gas meter (M ) model BK-G6 (Elster GmbH, Germany) meas- This correction factor k is calculated according to ges ured the total volume flow ( ) including the inlet air by BGZ Eq. (3) and refers to the total volume measured by the ane- leakage before and after the filter holder. To provide a wide mometer but not passing through the filter. range of different volume flows for each filter, a side chan- nel blower model SKV ND-150-3-935 (SKV-tec GmbH, k = k × k . ges Rest FL (3) Germany) was used in vacuum mode. At last, for the deter- The change of condition between in- and outlet of the mination of an alternative measurement method, a pressure sample, can be modelled as isothermal. Therefore, at atmos- measurement device was placed at the pipe connecter (M ). pheric condition, a pressure drop of 40 mbar corresponds to The determination of the volume flow by means of speed a maximum error of 4% (according to ideal gas equation) measurement by a hydrometric vane took place via the equa- of the measured volume flow with the vane anemometer. tion of continuity (1). As mentioned, many other factors (air leakage, grid, turbu- V = v × A, (1) FA FA lences etc.) influence the vane anemometer signal. All fac- tors are summarized by the correction factor k which was ges v denotes the gas velocity measured by hydrometric vane. FA empirically determined (Fig. 2). This factor is only valuable In Fig. 1B this point is marked by the circle M It can be for the used filters and the tested range of volume flow. By assumed that the volume flow, which is determined by correcting the filter surface and the air stream not passing means of the speed of the vane anemometer and the filter through the filter but through the system, the determination surface (A), is higher than the volume flow actually flowing of the real volume flow V is now possible by means of FA.k through the filter: gas velocity measurement. To stabilize the filter during sampling, a grid was inserted Distinct types of membrane filter discs commonly used into the filter carrier. The diameter of the grid and the inner for air-based sampling in occupational hygiene assessment diameter of the ring are the same size (40 mm). However, the (Soo et al. 2016) were used for the physical characterization grid reduces the effective filter area and this fact is consid- of the newly developed filter holders. All filters had a diam- ered by modification of the equation used to determine the eter of 47 mm to fit into the filter holder and included the Fig. 2 Diagram showing the relationship between air stream veloc- holder was according to Fig.  1 and for details on the used filters see ity and the correction factor k with different filter materials, n = 28. descriptions in Materials and Methods ges Experimental setup of establishing the correction factors of the filter 1 3 408 Aerosol Science and Engineering (2021) 5:404–418 following filter types: acrylic copolymer membrane filters in a C-Chip Haemocytometer (Neubauer improved; Incyto, Versapor 1200 (pore size 1.2 µm, Pall Inc. product #66394) South Korea) disposable counting chambers. All spores in and Versapor 3000 (pore size 3  µm, Pall Inc. product the nine big squares were counted using a microscope and #66387), mixed cellulose ester/cellulose nitrate membrane a 400-fold magnification, the mean and final concentration filters GN-4 (pore size 0.8 µm, Pall Inc. product # 64679) were calculated. Homogenous spore suspensions (2.0 × 10 and GN-6 (pore size 0.45 µm, Pall Inc. product # 66191), spores/mL) of T. longibrachiatum in 0.01% Tween 20 were hydrophilic polycarbonate membrane filters Isopore (pore aerosolized and subsequently sampled with the test devices. size 0.4 µm, Merck-Millipore product # HTTP04700), pol- ytetrafluoroethylene (PTFE) membrane filters type TF1000 2.3.2 Cultivation (pore size 1 µm, Pall Inc. product # 66155), type Zefluor 0.45 (pore size 0.45 µm, Pall Inc. product # P5PQ047), and For sampling in the bioaerosol chamber modified HYCON- type Zefluor 2 (pore size 2 µm, Pall Inc. product #P5PJ047). YM medium (RCS-T; peptone 6 g/L; D (+)-glucose mono- hydrate 15  g/L; malt extract 5  g/L; yeast extract 1  g/L; 2.3 Air Sampling agar–agar 16 g/L; pH 7.3 ± 0.2; 0.1% Triton™ X-100) was used. For the field study, microbial particles were cultivated The newly developed filter system was tested under con- on malt extract agar (MEA; Merck, United States); MEA trolled conditions in a bioaerosol test chamber (Pogner et al. containing Rose Bengal (MEA-BR; 25 mg/L, respectively, 2019) and during a field study with different indoor mold 50  mg/L Rose Bengal); dichloran glycerol agar (DG18; situations. In both cases the MAS-100 NT impactor was Merck, United States); and RCS-T medium. No significant used as a reference device for testing the suitability of the differences were detected for the tested Rose Bengal concen- novel filter holders for bioaerosol sampling. trations (data not shown). Colony forming units (CFUs) were counted after 6–7 days of incubation at room temperature 2.3.1 Bioaerosol Chamber (22 °C). CFUs were converted to CFU per m sampled air (CFU/m ). The sampling conditions in the bioaerosols chamber and the according experimental setup used for this study are based 2.3.3 Impaction Sampling on Pogner et al. 2019. In short, the test system that was used for the biological validation of the sampling device, Air sampling with the MAS-100 NT (MAS) (MBV, Swit- comprised of the CCB 3000 bioaerosol chamber (Calibra- zerland) was performed with a constant airflow of 100 L/ tion Chamber for Bioaerosols 3000, Palas, Germany) and min for 2 min for the bioaerosol chamber and field study an LSA nebulizer (Liquid Sparging Aerosolizer, CH Tech- sampling. 18 replicates were conducted for the bioaerosol nologies, United States). The bioaerosol chamber features a chamber experiment. For the field study 4–6 replicates were laminar airflow, even particle distribution and a testing area taken per sampling campaign, site, and medium. A statistical of 0.57  m . The MAS-100 NT and the filter holder of the correction of the impactor was not needed, since CFUs were air filtration-based system were placed in the chamber. The well within the countable range (Ghosh et al. 2015; Mandal casing of the vacuum cleaner was placed outside and was and Brandl 2011). connected to the filter holder through a tube connection in the chamber wall. For the spore suspensions, fungal strains 2.3.4 Filtration‑Based Microbial Sampling were cultivated on malt extract agar (Merck, United States) for 2–3 weeks at room temperature (22 °C). Trichoderma The filter system combined with a vacuum cleaner (VAC) longibrachiatum was used as test organism, since it was used was used for air filtration sampling with different mem- to validate the CCB 3000 bioaerosol chamber as described in brane filter types commonly employed in bioaerosol sam- Pogner et al. 2019 and its conidia can be aerosolized without pling: Versapor 1200 acrylic copolymer membrane disc detergent. As described previously, the use of detergents in filters (V 1200; 47 mm, pore size 1.2 µm, Pall Inc., United aerosolization creates a high load of unspecific particle back - States), cellulose nitrate/mixed cellulose ester filters (GN- ground and thus disqualifies parallel control measurements 6, 47 mm, pore size 0,45 µm, both from Pall Inc., United with the particle counter installed inside the chamber. Har- States), and polycarbonate filters (PC; Isopore membrane, vesting of the spores was performed by taking mycelia and 47 mm, pore size 0.4 µm, Merck-Millipore, Germany). Air spores from the plate with a spatula, adding 0.01% Tween filtration-based sampling for the field study was performed 20 to the plates, vortexing and filtering the suspensions with Versapor 1200 filters (V 1200). A series of different air through sterilized glass wool to remove mycelial cells. The velocities was measured with an impeller anemometer (air- spore suspension’s concentrations (given in spores/mL) were flow meter PCE-TA 30, PCE Instruments, Germany) for all determined by counting a 10 µL aliquot of a proper dilution filters used in this study and according air sampling (L/min) 1 3 Aerosol Science and Engineering (2021) 5:404–418 409 were established (Table ST1). By measuring the air velocity on the media, the sampling device or a combination of these (m/s) of each vacuum cleaner prior to air sampling, the cor- factors. responding sampling volume can be calculated according to Table S1 independent from the performance and type of the 2.3.6 Field Study Fungal Community Spectra vacuum cleaner. For the bioaerosol chamber measurements a “Siemens Super XS dino e, 2200 W” vacuum cleaner (Sie- For the field study, CFUs were counted and classified mens, Germany) was used. Based on the same air velocity according to the groups Alternaria, Cladosporium, Asper- of the vacuum cleaner (0.89 m/s), sampling was performed gillus/Penicillium, Aureobasidium, Fusarium, Neurospora, for 3 min for each of the tested filters (Versapor 1200 fil- Rhizopus/Mucor, Trichoderma, Chaetomium, Epicoccum, ter, Polycarbonate filter, and Cellulose Nitrate filter). Dur - Botrytis, and yeast. The selection of these taxa was based ing the field study sampling was conducted with Versapor on literature indicating frequently occurring outdoor and 1200 filters (V 1200) and a “Simpex 17730, 800 W” (Sim- indoor airborne fungal genera (Bowers et al. 2013; Fierer pex, United States) vacuum cleaner for 3 min 20 s at an air et  al. 2008; Hyvärinen et al. 2001; Lymperopoulou et al. velocity of 0.80 m/s. Five to 6 replicates on 2 independent 2016; Yamamoto et al. 2012). To enable a fast and efficient sampling days were sampled during for the bioaerosol cham- analytical evaluation, the taxa Aspergillus/Penicillium and ber experiments. Two replicates on 4 independent sampling Rhizopus/Mucor were combined in 1 classification due to campaigns were taken per sampling site and medium during their high morphological similarities, as suggested by other the field study. Previously determined correction factors for studies (Li and Kendrick 1995). Unidentified fungal colonies each filter material were considered in the calculations (see were classified in the categories "other sporulating fungi" Sect. 2.2). and sterile mycelia. Rankings were determined correspond- ing to the relative abundance within the sample (1 = low 2.3.5 Field Study Sampling Sites abundance; 2 = medium abundance; 3 = dominant). A taxon was considered as dominant if CFUs for that community The field study was conducted at an industrial unit of a spar - fraction exceeded 75% of the total cell counts. Subsequently, kling wine factory. Further details on the sampling site are low and medium abundances were defined accordingly. Data described in Unterwurzacher et al. (2018). The sampling were illustrated in a heat map showing relative abundances sites covered a wine cellar with obvious mold growth, an within one sample. Technical replicates of each sampling office that is primarily used as storage room without indica- day, sampling location, and cultivation medium were tions of mold infestation and an inner courtyard that sepa- combined. rated the indoor sampling sites and served as outdoor refer- ence. During sampling, a minimum distance of 1 m was 2.4 Statistical Analysis and Linear Regression kept between each sampling device and any other object Models (including other sampling devices, walls, and/or person- nel). Relative humidity and temperature were monitored. Statistical analyses were conducted using R version 3.2.4 (R The observed relative humidity and temperature ranges are Core Team 2015). Pearson’s correlation coefficients between listed in Table ST 2. Four independent sampling campaigns sampling devices and environmental factors (temperature were performed between June 2015 and January 2016. In and relative humidity) were calculated using cor.test (x, y). detail, sampling was conducted at 23 June 2015 (sampling Pearson’s coefficients were used in respect to creating first 1), 5 October 2015 (sampling 2), 23 November 2015 (sam- order linear models and avoiding overfitting of these models pling 3) and 27 January 2016 (sampling 4). for comparison between different sampling devices. Normal Besides the wine cellar with obvious mold growth, an distribution of the residuals was tested by the Shapiro–Wilk indoor sampling site (office) without indications of mold test and p values less than 0.05 were considered significant. infestation was chosen to represent both, a highly contami- For the comparison of different sampling devices, multi- nated site as well as a sampling site with low levels of air- dimensional linear models were developed. These models borne mold particles. Outdoor samples were used as ref- consider besides the dier ff ent sampling methods (CFUs) also erence to determine background levels at the location and the factors temperature and relative humidity. The respective to check for distinct differences between the fungal com- coefficients for each factor were estimated by minimizing the munities indoors and outdoors. The MAS-100 NT impactor sum of squared residuals and the assumption that y-intercept (MAS) was used for validation of the results obtained by the is zero. For estimating the regression coefficients of linear newly designed filter holders. Furthermore, 4 different culti- models, the means of 5 technical repeats were considered. vation media were compared, to investigate if certain fungal For linear least squares regression computation, the R func- communities might be underrepresented, either dependent tion “lm” was used. 1 3 410 Aerosol Science and Engineering (2021) 5:404–418 Differences between mean CFU values of different media the false air entry V . The numerical results are listed in FL or sampling devices were calculated using the R function t Fig. S1 and represent the mean of three measurements, each test (x,y). p values less than 0.01 were considered signic fi ant. of it was determined at four different partial loads. The filters Standard deviations of measurements conducted during GN-6 and Isopore (PC) have a comparatively high correction the field study are shown as boxplot. In the boxplot the cal- factor k due to lower porosity and, therefore, a higher pres- FL culated upper and lower quartiles (75th and 25th percen- sure drop of the filter. Results are shown and summarized tile), the maximum and minimum values (upper and lower in Fig. 2. In summary, it was established that even with a whiskers), and the median (excluding outliers) are indicated. simple vacuum cleaner the adaptor system can be used and If data exceeded 1.5 times, the interquartile range of these correct measurements can be performed as air leakage is measurements were considered as outliers (circles). not critical factor for the novel bioaerosol collection system. 3.2 Sampling Device Comparison Under Controlled Conditions 3 Results A bioaerosol chamber was used to establish stable sampling 3.1 Filter System Characterization conditions for the reference sampler (MAS) and the air fil- tration-based system to be validated (VAC) as described To determine the correction factor k , the parameters k ges Rest before (Pogner et al. 2019). Three different filter materials and k were measured individually. For k , deter mination, FL Rest were tested with the air filtration-based sampler in this com- the filter attachment site with the ring fixing the filter and parative study under controlled conditions. Similar sampling the adaptor were glued airtight, so that it can be assumed ̇ concentrations between the distinct filter materials were that the air volume flow due to leakage V is equal to zero. FL detected (162–206 CFU/m ), with the exception of the com- For this experimental setup, k can be calculated using Eq. Rest ̇ parison of the Cellulose Nitrate and the Polycarbonate filter, (4), where v is the gas velocity at the filter inlet, V is FA BGZ where mean CFU/m values were in a comparable range, but the total volume flow, v denotes the velocity measured by FA differences were significant (p value < 0.01). Independently hydrometric vane and A is the filter surface area. from the tested l fi ter materials, the air l fi tration-based system showed highly significant impairment in sampling Tricho- BGZ k = . (4) Rest v × A derma longibrachiatum spores compared to the MAS-100 FA NT impactor (MAS), with on average 66.9% lower CFU The air passing through the system but not through the counts detected by the air filtration system (Fig.  3). Con- filter is termed “false air”. In our setup this results from the sidering the broad stable range of different air velocities difference between the volume flow of the diaphragm gas (roughly 0.7–2.0 m/s) tested during the establishment of meter V and the corrected volume flow of the hydromet- BGZ the filters’ correction factors (Fig. S1) and the comparable ric vane V according to Eqs. (5) and (6). FA.k sampling efficiency with the 2 other tested filter materials in the bioaerosol chamber, in further consequence the Versapor ̇ ̇ ̇ V − V = V , (5) BGZ FA.k FL 1200 filter was used for the field study. and, 3.3 Comparative Field Study FL = false air % . (6) BGZ For further validation of the air filtration-based sampler, a comparative e fi ld study was conducted at the production site The test series for determining the correction factor k , Rest of a sparkling wine factory during 4 independent sampling was carried out with airtight glued filter components. As campaigns. a result, the entry of false air V due to leaks in the filter FL attachment could be excluded and the volume flow of the diaphragm gas meter V can be considered as the actual 3.3.1 Detected Colony Forming Units BGZ o fl w rate through the l fi ter. To determine the false air portion of the filter system, the experiments for the determination of Independent from the sampling site and sampling device, k were repeated with non-bonded components. Identical there was a clear seasonal shift in airborne fungal particles Rest to the test series of the bonded components, the volume flow (Fig. 4). Overall highest CFU values (up to 2054 CFU/m 3 3 of each filter was plotted over the velocities at four different outdoors; 3196 CFU/m wine cellar; 724 CFU/m office) partial loads and a linear regression model was formed. The were measured at sampling campaigns 1 and 2. difference in the volume flows at the same speeds results in 1 3 Aerosol Science and Engineering (2021) 5:404–418 411 While cultivation medium had little influence on CFU counts from filtration-based sampling, signic fi ant die ff rences (p value < 0.01) were found for 41.7% of measurements from the MAS-100 NT (MAS) (Table ST3). Comparing differences in sampling devices, particularly outdoors the MAS-100 NT impactor (MAS) and the air fil- tration-based sampler detected fungal CFUs in a highly simi- lar range (MAS: 2054–230 CFU/m ; VAC: 1618–241 CFU/ m ). In the wine cellar, the air filtration-based sampler measured significantly higher CFU values compared to the MAS-100 NT impactor (MAS: 1731–463 CFU/m ; VAC: 3196 ± 365 CFU/m ). The CFU levels detected in the office were similar between the two sampling systems and ranged between MAS: 703 ± 48 CFU/m and VAC: 724 ± 98 CFU/ m . 3.3.2 Measurement Stability Based on Relative Standard Deviations Fig. 3 Results of the bioaerosol chamber experiments. Compari- Overall, sampling with the MAS-100 NT (MAS) showed son of CFU detection of Trichoderma longibrachiatum spores under a high reproducibility with slightly lower relative standard controlled conditions in a bioaerosol chamber using the filter-adaptor deviations (rsd) of the measurement standard deviations system. The MAS-100 NT (MAS) was used as a reference device compared to the air filtration-based sampler (median of rsd: (n = 18). Different membrane filters were tested for the air filtration- based sampling using the vacuum cleaner-adaptor system (VAC): MAS: 12.8%; VAC: 19.4%) (Fig. 5). Particularly outdoor VAC Versapor: Versapor 1200 filter (n = 5), VAC Cell.Nitr.: cellulose measurements conducted with the air filtration-based sam- nitrate GN-6 filter (n = 6), and VAC Polycarb: Isopore polycarbonate pler varied strongly, while MAS-100 NT data were more filter (n = 6). Asterisk indicates significant difference to MAS refer - robust (median of rsd: MAS: 12.1%; VAC: 24.1%). Highest ence sampler similarities in measurement reproducibility were found in the office with little expected spore counts in the aerosols, where both sampling devices showed relatively high data fluctuations (median of rsd: MAS: 18.4%; VAC: 22.4%). Fig. 4 Results of the field studies. CFU counts detected at a comparative field sampling study at four different sampling days (1–4) conducted with the MAS-100 NT (MAS) and air filtration (VAC; on Versapor 1200 filters) for the tested cultivation media DG18, MEA, MEA-BR, and RCS-T. Results are given for each sampling site. Asterisk indicates significant difference to MAS reference sampler 1 3 412 Aerosol Science and Engineering (2021) 5:404–418 Fig. 5 Measurement reproducibility of the air filtration system. Rela- Fig. 6 Correlations of detected CFU counts between the reference tive standard deviations [rsd (%)] of field samplings were calculated sampler MAS-100 NT (MAS) and the air filtration-based sampler from the technical replicates to the means of the sampling campaign (VAC; on Versapor 1200 filters). All sampling campaigns and culti- for the MAS-100 NT (MAS) and the air filtration-based sampler vation media are shown. Data are indicated dependent on the sam- (VAC; on Versapor 1200 filters). Boxplots include data from four pling locations of the comparative field study (outdoor reference; independent sampling campaigns and four different media, resulting office; wine cellar) and the continuous line represents the overall lin- in 16 data points for each sampling location and sampler. In the box- ear correlation plot the calculated upper and lower quartiles (75th and 25th percen- tile), the maximum and minimum values (upper and lower whiskers), the CFU counts from another device in this study, consid- and the median (excluding outliers) are indicated. If a given SD value exceeded 1.5 times the interquartile range, this value was considered ering environmental temperature and relative humidity: as outlier (circle). Values used for statistical analyses shown in the CFU counts(sampling device 1) boxplots are listed in Supplementary Table ST 4 = A × CFU counts(sampling device 2) CFU Contrary to that, both samplers displayed the lowest rela- + B × Temperature C temp. tive standard deviations in the wine cellar, which generally + C × relative humidity [%]. rel.hum. (7) exhibited high CFU levels due to the obvious indoor mold infestation (median of rsd: MAS: 9.3%; VAC: 14.6%). Besides CFU counts, only the influence of humidity 3.3.3 Sampler Correlations and Linear Regression Models was statistically significant. Temperature was of minor importance. The herein presented correlations are valid for A linear regression model for comparison of sampling this study. Further comparative measurement campaigns devices and methodologies based solely on CFU counts would be needed for more general correlations. Also, poly- showed already high comparability between the sampling nomial functions were investigated but could not bring a devices (MAS-100 NT and air filtration) with an R value significant improvement to our models (data not shown). of 0.90 (Fig. 6). Furthermore, Pearson’s correlations (over- Measurements taken outdoors could not sensibly be fitted all data Shapiro–Wilk test of normality, p value > 0.05) at each sampling site substantiated these strong correlations Table 1 Regression coefficients for linear models (Eq.  7) to compare between the compared air samplers (outdoors: cor = 0.96, sampling devices based on measured values in this comparative study p value < 0.01; cellar: cor = 0.89, p value < 0.01; and office: Sampling Regression coefficients Adj R p Shapiro– cor = 0.77, p value < 0.01). device Wilk Furthermore, we developed multi-dimensional linear A B C CFU Temp Humid models that implement also the factors of temperature VAC → MAS 1.026*** − 2.638 5.376*** 0.9473 0.939 and relative humidity (Supplementary Table ST2). Con- sidering these additional parameters, the adjusted R value Significance codes: ***0.001; **0.01; *0.05 reached nearly 0.95 (Table 1). Equation 7 allows calculat- a Shapiro–Wilk test of normality, p values > 0.05 indicating normality ing CFU counts from a specified device in our study given are highlighted in bold 1 3 Aerosol Science and Engineering (2021) 5:404–418 413 into these models as there may be influencing factors that medium. While outdoors especially Cladosporium and were not measured in our experiments (e.g., wind velocity Alternaria species were detected, in the wine cellar Aspergil- and air turbulence). lus/Penicillium species were dominant, indicating an indoor mold infestation. In the vacant office, besides typical outdoor 3.3.4 Culture‑Based Fungal Community Spectra taxa Cladosporium and Alternaria also Aspergillus/Penicil- lium species were detected more frequently, which could be Besides CFU values, the influence of cultivation media and explained by whirled up dust during sampling rather than by sampling devices on the detected fungal community spectra indoor mold growth. was investigated during the field study. To cover all possible Generally, fungal community compositions sampled with underrepresented fungal genera, 4 different sampling media the MAS-100 NT (MAS) impactor showed slightly higher (DG18; MEA; MEA-BR; RCS-T) with distinct cultivation diversity at all sampling locations. For example, colonies characteristics were chosen. Community composition was belonging to the fungal taxa Aureobasidium, Trichoderma, highly influenced by both, sampling method and growth and Epicoccum were almost exclusively detected with the Fig. 7 Community spectra of cultivated airborne microbial particles The heat map displays relative abundances of the fungal communities sampled with the MAS-100 NT (MAS) and the air filtration-based detected within the sample. The color code is indicated. Yellow: high- system (VAC) during the field study. All sampling days (1–4), sam- est abundance; dark blue: lowest abundance; black: below detection pling locations (outdoor reference; office; wine cellar) and different limit (bdl). Mean values of replicates are represented (color figure cultivation media (DG18, MEA, MEA-BR; RCS-T) are illustrated. online) 1 3 414 Aerosol Science and Engineering (2021) 5:404–418 MAS-100 NT (MAS). For the most dominant genera such NT control device as defined in our previous study (Pogner as Alternaria, Cladosporium, and Aspergillus/Penicillium et al., 2019). As outlined in the Sect.  2 Trichoderma was similar abundances were found with both sampling devices used because it is one of the few species whose spores can (Fig. 7). be aerosolized without detergent and thus allows parallel control measurements with a particle counter installed inside the chamber. Moreover, in the subsequent field sampling 4 Discussion comparative studies were broad in terms of conditions, loca- tions, and expectable fungal biodiversity. Here, we present the development and evaluation of a new Generally, the testing in the bioaerosol chamber demon- filter-based bioaerosol sampling system that can be operated strated measurement stabilities of viable T. longibrachia- with commercially available vacuum cleaners as air pumps. tum spores similar to values reached by the MAS-100 NT, It opens the possibility to perform multiple sampling cam- suggesting high detection reproducibility of the novel air paigns in parallel in a short period of time at many die ff rent, filtration system under conditions without wind effects. In geographically distant locations. Such samplings are done future, more microbial species will be compared between for instance for epidemiologic studies to screen pathogens the filter-based sampler and a reference sampler in the bio- or monitor antibiotic resistance genes by molecular biol- aerosol chamber to gain better insights into possible bias for ogy analysis. Until now, large-scale comparative samplings certain types of microbes. are not routinely performed because at each of these distant As reported by Elmashae and coworkers (Elmashae et al. sampling locations, a standard bioaerosol sampler is needed 2017), which tested different filter materials accordingly to and the associated costs usually exceed the budget available identify airborne bacterial cells, our results showed similar for such studies. The adaptor and sterile filter unit composite detection rates with all tested filter materials. It has to be serve as a feasible option for routine analytics as the system mentioned, that pore sizes of the different membrane fil- is highly cost-effective compared to other sampling devices, ter discs tested in the bioaerosol chamber were not identi- since beside a vacuum cleaner that is usually available and cal, but a similar size range (Versapor 1.2 µm pore size; an anemometer, no further equipment is required. The phys- Cellulose Nitrate and Polycarbonate 0.8  µm pore sizes). ical tests of the sampling system with different air filters Nevertheless, considering that typical fungal spores feature resulted in the formulation of a correction factor for each sizes from approx. 1–30 µm (Yoo et al. 2017), the effect of filter to be applied. This factor in combination with a simple the different pore sizes is expected to be rather small. This anemometer gave consistent sampling volumes in each of the would be in agreement with the findings of Soo et al. ( 2016), tests run. Therefore, the combination of the filter-adaptor who detected similar particle collection efficiencies in the and vacuum cleaner can thus be regarded as reliable bio- range of 1 µm pore sizes and strong deviations only com- aerosol sampling system. An uncertainty of the system that pared to higher pore sizes among different commonly used has so not been tested in the long run may be a reduction of air filter materials. vacuum generation by the vacuum cleaner. The performance The new filter system was also functional in displaying of the system might suffer from reduced electric engine per - expected seasonal fluctuations in cultivatable fungal cells. formance after a long sampling campaign. However, field It is well known that the composition and biodiversity of data from more than 300 individual test runs as presented airborne microbial cells as well as their concentrations here gave quite consistent CFU counts in comparison to the change strongly with seasons (Bowers et al. 2013; Frankel reference sampler and thus we do not foresee large problems et al. 2012a; Heo et al. 2014; Matthias-Maser et al. 2000). due to reduction of performance of the vacuum cleaner used As reported before by Bowers and colleagues (Bowers et al. for sampling. 2013), also our results showed highest fungal quantities Although the biological sampling efficiency was com- between summer and fall and lowest in winter. The diverse parable to the reference device in our field studies, under sampling time points covering different seasons were cho- controlled conditions in a bioaerosol chamber, the air filtra- sen for this study to determine if not only number but also tion-based sampler showed significantly lower CFU-based biodiversity of monitored microbes correlate between our collection efficiency of aerosolized Trichoderma longibra- device and the reference sampler. Strong overall correlations chiatum spores. The susceptibility of the sampled micro- between the tested and the reference devices indicate high organism due to diverging sampling stress pursued by the measurement reliability of the new filter holder system for die ff rent sampling principles might explain this discrepancy environmental samples (Ghosh et al. 2015; Kalogerakis et al. (Wang et al. 1999). Although a variety of different species 2005; Yao and Mainelis 2006). could have been tested with the new filter system in the To investigate if certain fungal species are underrepre- chamber, we decided to perform this first step in validation sented in the newly developed sampling system, four dif- only under the most suitable conditions for our MAS-100 ferent cultivation media were compared during the field 1 3 Aerosol Science and Engineering (2021) 5:404–418 415 study. The tested cultivation media covered highly divergent filtration system. Higher susceptibility to desiccation stress growth properties including different carbon and nitrogen during air filtration might explain the underrepresentation sources, water activities and inhibitory compounds. The air of more desiccation-sensitive species (Ghosh et al. 2015; filters detected comparable CFU counts on all tested culti- Wang et al. 2001). vation media, which was not the case for the MAS-100 NT. Overall, the field study indicated a strong and highly sig- A reduction in biodiversity due to higher sampling stress nificant correlation between the newly designed filter holders (Zhen et al. 2018) during filtration (see below) could explain and the well-established impactor system. The implemen- the minor influence of cultivation media on enumeration of tation of multi-dimensional linear models for the indoor fungal propagules. Fungi resistant to the stress imposed by measurements considered the environmental factors tem- filtration probably grow equally well on different growth perature and relative humidity, which further strengthened media. the correlation. Given the results from these models, it can There was also no significant bias towards indoor or be deduced that especially relative humidity has a significant outdoor locations. Particle counts detected outdoors and in impact on the comparability between the different sampling the office were in a highly similar range for both sampling methods used in this study. The temperature did not show a devices. In the wine cellar, particularly at sampling cam- significant influence on the measurement correlations. Both paigns 1 and 2, the filter-based system reached higher CFU bioaerosol samplers were subjected to some species related counts compared to the MAS-100 NT. Since the community restrictions in detecting viable fungal cells, but overall the spectra of the office and the outdoor reference are more alike CFU-based results were comparable and the CFU regression as shown in another study conducted at this site (Unterwur- coefficient of 1.026 (p value < 0.01) indicates the reproduc- zacher et al. 2018), discrepancies in particle detections in ibility of the compared sampling devices during environ- the wine cellar might be again a result of species related mental conditions. underrepresentation. Obviously, presented results are valid for the sampling However, one weakness of the newly developed system settings chosen in this study, different sampling durations might be the higher variability of detected fungal counts. or airflow rates might lead to diverging correlations, since Overall, relative standard deviations were higher for the air sampling settings are crucial for the overall performance of filters compared to the impactor at the chosen conditions bioaerosol samplers (Grinshpun et al. 2007; Han et al. 2015; and experimental design, i.e., two technical replicates per Uhrbrand et al. 2017). Although a broad range of environ- cultivation media, per sampling site, and per sampling date. mental conditions, including different seasons, indoor sam- Although this sampling strategy still concurs with official pling sites with high, respectively, low fungal particle levels directives and guidelines, e.g., DIN ISO 16000-16 (2008) and outdoor reference measurements were covered within the comparatively low number of repeats might explain this experimental setup, further studies at different sampling higher relative standard deviations. Furthermore, filters need sites are needed to verify these finding. One limitation of additional manipulation steps after sampling, while plates the herein presented field study is the rather small number from the MAS-100 NT impactor are directly incubated after of technical replicates, which possibly explains the higher sampling. Handling of filters can potentially increase varia- relative standard deviations compared to the MAS-100 NT tion in enumeration of fungal propagules. reference device. Similar to other comparative studies of bioaerosol sam- plers, we also found differences in community composition depending on the sampling device (Lukaszuk et al. 2017; 5 Conclusion Unterwurzacher 2016). Nevertheless, it cannot be excluded that particle dispersal not only occurred through the overall The findings underline the applicability of the novel vacuum- sampling activity (Meadow et al. 2014) but especially dur- cleaner-based filter sampling system to detect viable fungal ing the use of the vacuum cleaner (Corsi et al. 2008; Knibbs cells and spores in bioaerosols. The system is largely equiva- et al. 2012). lent to other commonly used samplers as it gave similar results Sampling with the MAS-100 NT impactor showed as the reference MAS-100 NT impaction sampler. The advan- slightly higher diversity at all investigated sites. Although tage of this system is that (1) no extra air pump or collector most dominant taxa including Alternaria, Cladosporium, needs to be purchased as vacuum cleaners are usually avail- and Aspergillus/Penicillium were detected in a similar range, able, (2) materials used for collection (sterile filters) endure other taxa such as Aureobasidium, Trichoderma, and Epico- harsh temperature conditions during storage and thus have ccum were almost exclusively detected with the MAS-100 a long shelf life, and (3) the simplicity of the system allows NT impactor. This is consistent with the findings in the bio- large number sampling campaigns to be run in parallel at very aerosol chamber experiment, where Trichoderma longibra- distant locations (e.g., broad antibiotic resistance sampling chiatum spores were strongly underrepresented by the air campaigns). A weakness of the system may be the need of 1 3 416 Aerosol Science and Engineering (2021) 5:404–418 as you give appropriate credit to the original author(s) and the source, an anemometer to calibrate the air volume sampled per time provide a link to the Creative Commons licence, and indicate if changes unit, a possible underrepresentation of some genera depend- were made. The images or other third party material in this article are ing on the chosen filters and a slightly higher variability of included in the article’s Creative Commons licence, unless indicated the obtained results due to changing power capacities of the otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not vacuum cleaners. However, these slight disadvantages may be permitted by statutory regulation or exceeds the permitted use, you will compensated by opening the possibility to perform large paral- need to obtain permission directly from the copyright holder. To view a lel sampling campaigns at distant locations. Such scientifically copy of this licence, visit http://cr eativ ecommons. or g/licen ses/ b y/4.0/ . valuable endeavors would not be economically feasible if at each of these locations a costly commercial sampler needs to be purchased. Other bioaerosol compounds such as spore- References forming bacteria or pollen can equally be sampled with the described system once validation studies with these compo- Aizenberg V, Reponen T, Grinshpun SA, Willeke K (2000) Perfor- mance of Air-O-Cell, Burkard, and Button Samplers for total nents have been performed similar to the work presented here. enumeration of airborne spores. Am Ind Hyg Assoc J 61(6):855– 864. https:// doi. or g/ 10. 1202/ 0002- 8894(2000) 061% 3c0855: Supplementary Information The online version contains supplemen- POAOCB% 3e2.0. CO;2 tary material available at https://doi. or g/10. 1007/ s41810- 021- 00110-9 . An HR, Mainelis G, Yao M (2004) Evaluation of a high-volume port- able bioaerosol sampler in laboratory and field environments. Acknowledgements The authors wish to express their gratitude to Indoor Air 14(6):385–393. https:// doi. org/ 10. 1111/j. 1600- 0668. Dragana Bandian from Austrian Institute of Technology GmbH (AIT) 2004. 00257.x and the AQA team for technical support and sampling assistance. This Bernstein JA, Alexis N, Bacchus H, Bernstein IL, Fritz P, Horner E, work was supported by the Lower Austria Corporation for Research Li N, Mason S, Nel A, Oullette J, Reijula K, Reponen T, Seltzer and Education (NFB; Grant LS12-011) and by the DGUV together with J, Smith A, Tarlo SM (2008) The health effects of nonindustrial the AUVA (Grant FF-FP-0337). Special thanks to the occupants of the indoor air pollution. J Allergy Clin Immunol 121(3):585–591. site, where sampling was conducted. https:// doi. org/ 10. 1016/j. jaci. 2007. 10. 045 Bowers RM, Clements N, Emerson JB, Wiedinmyer C, Hannigan Author contributions Study design: SSG, MG, SB, CP, GT, and JS. MP, Fierer N (2013) Seasonal variability in bacterial and fungal Performed research: VU, MB, CP, AK, and HB. Analysis of data: SB, diversity of the near-surface atmosphere. Environ Sci Technol GT, HB, SSG, CP, JS, and MG. Writing of manuscript: VU, SSG, CP, 47(21):12097–12106. https:// doi. org/ 10. 1021/ es402 970s GT, HB, JS, and MG. All authors read and approved the manuscript. Burton NC, Grinshpun SA, Reponen T (2007) Physical collection effi - ciency of lt fi er materials for bacteria and viruses. Ann Occup Hyg 51(2):143–151. https:// doi. org/ 10. 1093/ annhyg/ mel073 Funding Open access funding provided by University of Natural Chang CW, Hwang YH, Grinshpun SA, Macher JM, Willeke K (1994) Resources and Life Sciences Vienna (BOKU). This work was sup- Evaluation of counting error due to colony masking in bioaerosol ported by the Lower Austria Corporation for Research and Education sampling. Appl Environ Microbiol 60:3732–3738 (NFB; Grant LS12-011) and by the DGUV together with the AUVA Corsi RL, Siegel JA, Chiang C (2008) Particle resuspension during the (Grant FF-FP-0337). use of vacuum cleaners on residential carpet. J Occup Environ Hyg 5(4):232–238. https://doi. or g/10. 1080/ 15459 62080 19011 65 Code availability Not applicable. Dannemiller KC, Gent JF, Leaderer BP, Peccia J (2016) Influence of housing characteristics on bacterial and fungal communities in Declarations homes of asthmatic children. Indoor Air 26(2):179–192. https:// doi. org/ 10. 1111/ ina. 12205 Deacon LJ, Pankhurst LJ, Drew GH, Hayes ET, Jackson S, Longhurst Conflict of interest The authors declare a potential conflict of interest PJ, Longhurst JWS, Liu J, Pollard SJT, Tyrrel SF (2009) Par- as a member of the company that may in future bring the sampling ticle size distribution of airborne Aspergillus fumigatus spores system to market is a coauthor of this manuscript. All authors declare emitted from compost using membrane filtration. Atmos Environ that this fact, however, has not influenced the design or interpretation 43:5698–5701. https:// doi. org/ 10. 1016/j. atmos env. 2009. 07. 042 of the results and that the research was conducted in the absence of any DIN-ISO16000-16 (2008) Indoor air. Part 16: detection and enumera- commercial or financial interest. tion of moulds—sampling by filtration, vol 16000-16. Interna- tional Standards Organization Ethics approval Not applicable. DIN-ISO13205-1:2014 (2014) Workplace atmospheres-Assessment of performance of instruments for measurement of airborne particle Consent to participate Not applicable. concentrations German Version. International Standards Dybwad M, Skogan G, Blatny JM (2014) Comparative testing and Consent for publication Not applicable. evaluation of nine different air samplers: end-to-end sampling efficiencies as specific performance measurements for bioaerosol Research involving human and animal participants This article does applications. 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Journal

"Aerosol Science and Engineering"Springer Journals

Published: Dec 1, 2021

Keywords: Bioaerosol sampling; Air filtration; Sampling device validation; Field study; Bioaerosol chamber; Indoor mold assessment

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