The goal of this research is to determine the human‐associated emission rates of bacteria and fungi in an occupied classroom. Particle size distributions of total airborne particulate matter, bacterial genomes, and fungal genomes (based on rRNA‐encoding gene copy numbers) were measured under occupied and vacant conditions, and a material balance model was applied to determine the per person emission rates of bacterial and fungal size‐fractionated particles attributable to occupancy. Bacterial phylogenetic libraries were produced for aerosols sampled under occupied conditions, and the size‐resolved emissions of human‐associated bacterial taxa were estimated. The size‐resolved emission rates and bacterial population characterization produced here represent an important step toward applying models to make inferences about the distribution of and human exposure to bacteria and fungi in occupied indoor settings.

Materials and methods

Test environment A university classroom located on the ground floor of a five‐story building in the northeastern United States was selected as the test room. This location was selected because of consistent and readily characterized occupancy levels, along with proximity to the research team and relative ease of securing access. Experiments were conducted in the fall of 2009 and included 4 days when the classroom and building were occupied, and 4 days when the classroom and building were vacant. On occupied days, the room was frequently accessed with three to five classes per weekday. For the four test days when the room was occupied, the room held an average of 4.7 people during a total of 22.2 h of sampling for a cumulative occupancy of 104.2 person hours. Classes were conducted in a lecture format. Students typically sat in desks and the instructor stood or moved throughout the front half of the classroom. Although the floor is vacuumed regularly, no cleaning was conducted at least 1 day before or during the experimental days. The volume of the classroom is approximately 90 m3 (L = 6.0 m, W = 5.0 m, H = 3.0 m), and the entire floor area was covered by lightly worn commercial medium pile level‐loop carpet. No mold or moisture problems have been reported for this building, and none have been observed during sampling. The outdoor environment near the building was highly vegetated and consisted of a tree‐lined street with lawn and flower gardens. There were no green plants in the room, nor were they common throughout the building. During the occupied and vacant experiments, all windows and doors were closed and conditioned air was delivered by the building HVAC system to the room through a 0.9 × 0.05 m air register situated above the door. Ventilation exhaust ports were located along the floor and near the wall opposite to the ventilation air inlet. Based on six deliberate CO 2 releases and tracer gas studies performed prior to air sampling and under vacant, well‐mixed conditions, the average air‐exchange rate (AER) ± s.d. was 5.5 ± 1.3/h. AER was also calculated immediately after occupancy based on the decay of exhaled CO 2 from occupants. This AER averaged 6.2 ± 0.9/h, which is not statistically different than the AER derived from tracer gas studies. During the four occupied and four vacant experimental days, the temperature was 23.5 ± 1.1°C indoors and 13.4 ± 2.4°C outdoors, and the average relative humidity was 28 ± 7% indoors and 45 ± 16% outdoors. The reported air‐exchange rate is in the upper range for ventilated commercial buildings (Persily et al., 2006). The average occupant density of 4.7 persons in 30 m2 (16 persons per 100 m2) is within ranges common for many indoor spaces with moderately dense occupancy (ASHRAE, 2005).

Sampling Both indoor and outdoor aerosols were sampled during the four occupied and four unoccupied experimental days. For indoor and outdoor size‐distributed samples, particles were collected on uncoated polycarbonate track‐etched filters (PCTE) that were loaded onto an 8‐stage non‐viable impactor (New Star Environmental Inc., Roswell, GA, USA) operated at a flow rate of 28.3 l/min. To increase the mass of collected material, six stages of the impactor were used with the following nominal cut‐points: 9.0, 4.7, 3.3, 2.1, 1.1, and 0.4 μm. An upper cut‐point for the initial stage was assumed to be 20 μm, based on the rapid removal of particles greater than 20 μm from indoor air owing to gravitational settling. To obtain genome copies above detection levels (Hospodsky et al., 2010) on all stages, the non‐viable impactors sampled air cumulatively for the four consecutive occupied or vacant experimental days. Between experimental days, the impactors were wrapped in autoclaved aluminum foil and stored at 4°C to inhibit bacterial and fungal growth. In addition to these impactor samplers, semi‐continuous particle counts were recorded indoors and outdoors on occupied and vacant days using optical particle counters with the following size channels: 0.3–0.5, 0.5–1.0, 1.0–2.5, 2.5–5.0, and 5.0–10.0 μm (MET ONE HHPC‐6, Hach Ultra Analytics Inc., Loveland, CO, USA).

Quantification of size‐resolved particle mass, bacterial genome copies, and fungal genome copies To determine particulate matter mass concentrations from impactors, filters were equilibrated and weighed before and after sampling. Weighing was performed using a precision balance (Mettler Toledo type AG245, Columbus, OH, USA). Static electricity was removed with a polonium αparticle source (Staticmaster static eliminator, NRD, Grand Island, NY, USA), and filters were equilibrated prior to weighing at constant temperature and humidity (30 ± 0.5°C, 32 ± 1.5% RH) for at least 24 h before weighing. Weighing was performed in triplicate and averages reported. The quantification of bacterial 16S rRNA‐encoding genes and fungal 18S rRNA‐encoding genes in all impactor stages was completed using real‐time PCR in accordance with previously developed protocols for DNA extraction, amplification, and calibration (Boreson et al., 2004; Hospodsky et al., 2010). Briefly, DNA was extracted from one‐quarter of the PCTE filter using a method that included cell lysis by enzymatic treatment and physical disruption through bead beating, phenol/chloroform isoamyl alcohol‐based isolation of nucleic acids, and DNA cleanup and concentration using spin columns from the MO BIO PowerMax Soil DNA kit (MO BIO, Inc., Carlsbad, CA, USA) (Boreson et al., 2004). Exceptions to the cited method included proteinase K incubation at 54°C instead of 37°C, omitting the freeze–thawing cycle during DNA extraction, and omitting the 1‐h, 65°C incubation step prior to beadbeating. Quantitative PCR was performed in triplicate using an ABI 7500 fast real‐time PCR system (Applied Biosystems, Carlsbad, CA, USA). For bacteria, universal bacterial primers and TaqMan® probes covered the 331 to 797 E. coli numbering region of the 16S rRNA‐encoding gene with forward primer 5′‐TCCTACGGGAGGCAGCAGT‐3′, reverse primer 5′‐GGACTACCAGGGTATCTAATCCTGTT‐3′, and probe (6‐FAM)‐5′‐CGTATTACCGCGGCTGCTGGCAC‐3′‐(BHQ‐1) (Nadkarni et al., 2002). For the universal fungal DNA quantification, a SybrGreen assay was used with primers reported by Zhou et al. (2000). These included the forward primers FF2 (5′‐GGTTCTATTTTGTTGGTTTCTA‐3′) and reverse primer FR1 (5′‐CTCTCAATCTGTCAATCCTTATT‐3′). Real‐time PCR standard curves of genome quantity vs. cycle threshold number for bacteria and fungi were developed using known amounts of Bacillus atrophaeus (ATCC 49337) and Aspergillus fumigatus (ATCC 34506) genomic DNA, respectively. To produce standard curves, three to five independent dilution series were produced corresponding to 101 to 106 genome copies for each organism. For bacteria, cycle threshold values were calibrated vs. total bacterial genomes and accounted for the ten rRNA operon copies in B. atrophaeus and the average of four rRNA operon copies per genome for all bacteria (Lee et al., 2009). The rRNA operon copy numbers in fungi are highly variable, even within species, and are not well cataloged. Thus, fungal universal qPCR values are presented as A. fumigatus genome equivalents by assuming 55 rRNA operon copies per genome for A. fumigatus (Herrera et al., 2009). To test for PCR matrix inhibition, standard curves for B. atrophaeus and A. fumigatus were also produced by spiking genomes into aerosol filter extract. No inhibitory effects were observed.

Emission rate estimation Total and microbial particle emission rates during occupancy were estimated by considering the room as a completely mixed flow‐through reactor in which the time‐averaged indoor concentration is the sum of two contributions: a fraction, f, of the time‐averaged outdoor concentration, plus a contribution from indoor emissions: (m1) In Equation (1), C is the time‐averaged indoor air concentration of total particle mass (μg/m3), bacterial genomes (genomes/m3), or fungal genomes (equivalent genomes/m3); C out is the time‐averaged corresponding outdoor concentration during monitoring, f is the indoor–outdoor ratio in the absence of indoor emissions (measured during the vacant sampling period); N is the average number of persons in the room during the occupied experimental time (persons); E is the per person emission rate of total particle mass (μg/h/person), bacterial genomes (genomes/h/person) or fungal genomes (equivalent genomes/h/person), Q is the volumetric ventilation rate (m3/h); V is the room volume (m3), and k is the size‐specific deposition‐rate coefficient for total particles, bacterial genomes, or equivalent fungal genomes (per h). The ventilation rate was estimated as the product of the room volume and the measured air‐exchange rate. Size‐specific deposition‐rate coefficients were derived from previously reported values (Thatcher et al., 2002) and are listed in Table 1. These specific rates were chosen because of their coverage of the super micron range (for 0.5–9 μm particles) and were extrapolated to the largest size section considered in the present work. Utilizing the impactor‐measured indoor and outdoor concentrations for the occupied and vacant sampling periods, Equation (1) was solved for the per person, size‐resolved emission rates (E) corresponding to total airborne particle mass, bacterial genomes, and equivalent fungal genomes. Table 1. Deposition‐rate coefficients (k) for each impactor size stage used to estimate particle mass, bacterial genome, and equivalent fungal genome emission ratesa 0.4–1.1 μm 1.1–2.1 μm 2.1–3.3 μm 3.3–4.7 μm 4.7–9 μm >9 μm Deposition loss‐rate coefficient (/h) 0.31 0.79 2.1 4.4 8.6 9.6