Airborne bacteria, fungi, and endotoxin levels in

Airborne bacteria, fungi, and endotoxin levels in residential microenvironments: a case study R. Balasubramanian, P. Nainar & A. Rajasekar

Aerobiologia International Journal of Aerobiology including the online journal `Physical Aerobiology' ISSN 0393-5965 Aerobiologia DOI 10.1007/s10453-011-9242-y

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Author's personal copy Aerobiologia DOI 10.1007/s10453-011-9242-y

ORIGINAL PAPER

Airborne bacteria, fungi, and endotoxin levels in residential microenvironments: a case study R. Balasubramanian • P. Nainar • A. Rajasekar

Received: 4 April 2011 / Accepted: 22 November 2011 Ó Springer Science+Business Media B.V. 2011

Abstract Limited data are currently available on the concentrations of airborne bacteria, fungi, and endotoxins in indoor environments. The levels of aerial bacteria and fungi were measured at several microenvironments within a well-ventilated residential apartment in Singapore including the living room, kitchen, bedroom, toilet, and at a workplace environment by sampling indoor air onto culture medium plates using the 6-stage Andersen sampler. Total microbial counts were determined by collecting the air samples in water with the Andersen sampler, staining the resultant extracts with a fluorescent dye, acridine orange, and counting the microbes using a fluorescent microscope. The levels of airborne endotoxins were also determined by sampling the airborne microorganisms onto 0.4 lm polycarbonate membrane filter using the MiniVol sampler at 5 l/min for 20 h with a PM2.5 cut-off device. The aerial bacterial and fungal concentrations were found to be in the ranges of 117–2,873 CFU/m3 and 160–1,897 CFU/m3, respectively. The total microbial levels ranged from 49,000

R. Balasubramanian (&) A. Rajasekar Department of Civil and Environmental Engineering, Faculty of Engineering, National University of Singapore, Block EA, 9 Engineering Drive 1, Singapore 117576, Singapore e-mail: [email protected]; [email protected] P. Nainar Department of Microbiology, Tirunelveli Medical College, Tirunelveli 627011, India

to 218,000 microbes/m3. The predominant fungi occurring in the apartment were Aspergillus and Penicillium while the predominant bacterial strains appeared to be Staphylococcus and Micrococcus. The average indoor endotoxin level was detectable in the range of 6–39 EU/m3. The amount of ventilation and the types of human activities carried out in the indoor environment appeared to be important factors affecting the level of these airborne biological contaminants. Keywords Bioaerosols Bacteria Fungi Airborne endotoxins Indoor Aerosols Allergens PM2.5

1 Introduction There is great concern about the potential health hazards of biological components in airborne particulate matter (bioaerosols), particularly about the levels of allergenic or toxigenic fungi and their association with indoor air quality (Zucker and Muller 2004; Liao et al. 2010; Hsu et al. 2011; Sykes et al. 2011; Nilsson et al. 2011; Park et al. 2011). The quality of indoor air is considered to be very important since an average individual spends up to 85–90% of time indoors (ASHRAE 1992). The occurrence of adverse health effects has been reported in several work environments (Lacey and Dutkiewicz 1994) such as agricultural (Heederik et al. 1991; Kullman et al. 1998) and

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Author's personal copy Aerobiologia

metal machining (Thorne and DeKoster 1996) settings, as well as in residential environments (Michel et al. 1991; Michel et al. 1996) due to the deterioration of air quality caused by bioaerosols. Bioaerosols are defined as airborne particles, large molecules or volatile materials that are either living, containing living organisms, or are released from living organisms (ACGIH 1989). Hence, they can exist as bacterial or fungal spores, bacterial cells or fragments of fungal mycelium, pollens, or microbial products such as endotoxins or mycotoxins (Reponen et al. 2001; Muilenberg 1995). These biological particles cover a large size range, from viruses, which are small at about 0.02–0.03 lm in length, to pollen, algae, protozoa, and dander, which are several tens to hundreds of micrometers in aerodynamic diameter (Reponen et al. 2001; Muilenberg 1995; Owen et al. 1992; Dowd and Maier 2000). They are often transported as attached to other particles, such as skin flakes, soil, dust, saliva or water droplets. Inhalation of some of these particles can cause irritations, infectious diseases, allergic and inflammatory reactions to the human respiratory system. Bioaerosols have been studied in numerous different regions and settings: schools (Aydogdu et al. 2005), child care centers (Zuraimi and Tham 2008), animal feed industry (Liao et al. 2010; Hameed et al. 2003), animal sheds (Rosas et al. 2001), rice mills (Savino and Caretta 1992; Desai and Ghosh 2003), saw mills (Platts-Mills et al. 2005; Oppliger et al. 2005; Jothish and Nayar 2004), food processing units (Zorman and Jersek 2008), bakeries and flour mills (Musk et al. 1989; Singh and Singh 1994; Awad 2007) hospitals, schools, senior care centers and nursing centers (Kim and Kim 2007) and social welfare houses (Hsu et al. 2011; Rolka et al. 2005). However, there are comparatively fewer studies available on levels of bioaerosols and their size distribution in residential indoor environments, where people spend a substantial time (e.g. Gorny et al. 1999; Shelton et al. 2002; Aydogdu et al. 2005; Lee and Jo 2006; Haas et al. 2007). These studies were carried out in a variety of indoor environments and showed a great variation in the total concentration of bioaerosols. However, particle size is critical with regard to their fate in the indoor air and their deposition in the human respiratory system. The size distribution of bioaerosols depends upon the type of micro-organisms (Reponen et al. 1994), the age of the spore and nutrient medium

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(Ellis 1981), relative humidity (Pasanen et al. 2000), differences in aggregation rates of the spores (Gorny et al. 1999), and the type of particles they are associated with such as mist or dust (Dowd and Maier 2000). Data are only sparsely available on the size distributions of bioaerosols and factors associated with them. Endotoxin, ubiquitous in the environment, is a biologically active lipopolysaccharide that is a component of the outer membrane of Gram-negative bacteria. Several epidemiologic investigations have shown a modest effect of endotoxin exposure on asthma morbidity (Michel et al. 1996). In humans, acute exposure to endotoxins can cause blood and lung inflammatory responses (Michel et al. 1992), resulting in respiratory symptoms such as shaking chills, fever, and severe asthma (Rennie et al. 2008; Park et al. 2000, 2001; Michel et al. 1992, 1996). In residential settings, house dust containing endotoxins could be an important determinant of asthma severity (Rylander et al. 1989; Michel et al. 1996; Gehring et al. 2002). Although some studies have suggested a protective role of endotoxin exposure in infancy, exposure to endotoxins in childhood and later in life appears to have a detrimental effect in both individuals with asthma and other respiratory conditions and in healthy volunteers (Michel et al. 1996; Schram-Bijkerk et al. 2005; Gehring et al. 2001; Thorne et al. 2009). Inhalation of airborne bacteria, fungi, and bacterial endotoxin has been recognized to be associated with numerous allergenic responses and respiratory symptoms. We hypothesize that the level of these airborne microbial organisms varies from one location to another within a home depending on the nature and intensity of human activities in specific microenvironments. It is therefore important to examine the level of bioaerosols in several locations within a residential home which will provide valuable information in the assessment of the quality of indoor air. With this motivation in mind, the present study was conducted in a tropical country with high temperature and humidity (Singapore) to investigate the total concentration and size distributions of culturable fungal and bacterial aerosols in several microenvironments of a residential home including the living hall, kitchen, bedroom, and toilet, and in a workplace environment. The relationship of the bioaerosol concentrations with relative humidity and temperature was examined. In addition, the bacterial endotoxins


levels present in particulate matter (PM2.5) were also estimated and are discussed in this article.

3 Materials and methods 3.1 Description of sampling sites

2 Background information Rising trends in mortality from asthma have been reported in many western countries including the United States (Sly 1988) and others (Rennie et al. 2008) as well as in Hong Kong (So et al. 1990). The sharpest increase in asthma deaths has been observed in the 5–34 year age group. In Singapore, an increase in asthma mortality was observed in children aged 5–14 years from 0.21 per 100,000 persons in 1970s to 0.72 per 100,000 persons in 1990s (Ng and Tan 1999; NUH 2002, 2003). Asthma is a major chronic health problem in Singapore because, on average, it afflicts one in 5 children and one in 20 adults (Lim 2003). Besides asthma, the high prevalence of children allergic diseases is also common in Singapore. Research findings on asthma and allergies by the Children’s Medical Institute (CMI), National University Hospital (NUH) in Singapore revealed that 20–21% of children suffered from asthma and 38–45% of children suffered from allergic rhinitis between 1994 and 2001. In comparison, only a mere 9.1% of children aged 6–12 years old were diagnosed with asthma in Seoul in 2000 (Lee 2010). Childhood asthma and allergic diseases are major health problems throughout the world. In many studies, it was shown that the prevalence is still increasing. A study in 2003 by the CMI, NUH and Department of Paediatrics, National University of Singapore (NUS) revealed that the symptoms of asthma and allergy are already prevalent by the second year of life in Singaporean children.

The indoor air samples were collected from various locations within a well-ventilated second-story apartment as well as from an air-conditioned office (i.e., workplace environment) in a different location to create several microenvironments; the latter was also chosen in this study to assess the exposure of individuals to bioaerosols in both home and workplace environments during the course of a typical work day. While the apartment, located in a multi-story building in a residential complex, was occupied by four persons and included a living room, a kitchen, two bathrooms, and three bedrooms, the office (located on the University NUS campus) was occupied by a single person (the lead author who carried out the study was the occupant of both the home and the office). The outdoor air samples were collected in the vicinity of the apartment (3 m away from the apartment). The details of the microenvironments sampled are listed in Table 1. 3.2 Microbiological air sampling Biological air samples were collected between December 2010 and February 2011 in triplicates at the two locations during the morning time (7 am) at both indoor and outdoor environments for 1 day per month. In the case of indoor air samples, concurrent bioaerosol measurements were made at two different sites within each location, i.e., bedrooms, kitchen, toilet, work place. For outdoor air, biological samples were collected in front of the apartment at a height of 1.5 m above the floor level, i.e., in the breathing zone of a standing person. In the indoor area (living room,

Table 1 Description of the sampling microenvironments Microenvironment

Abbrev.

Temperature (8C)

Relative humidity (%)

Bathroom

Bath

27.6–28.9

80–88

AC bedroom

AC bed

23.5–24.6

54–68

Non-AC bedroom 1

Bed

27.4–29.3

70–84

Non-AC bedroom 2

Bed act

27.4–29.3

70–84

Kitchen

Kit

27.2–29.0

69–88

Special activity

Air-conditioning at least 16 h daily Dust-creating activities changing bed linen, curtains

Outdoor

Out

26.6–31.3

62–92

Near vicinity of window of living room

AC office

Off

22.1–26.4

65–73

Air-conditioning at least 20 h daily

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bedroom, kitchen and toilet), air sampling was conducted at an approximate height of 1 m above the floor level, i.e., in the breathing zone of a seated person. Altogether, 76 biological air samples were collected in the both indoor and outdoor environments in all locations. Airborne bacteria and fungi were collected at each location by using an Andersen six stage cascade impactor operating at an air flow rate of 28.3 l/min for 10 min (Andersen 1958). A total of two air samples per microenvironment were collected for each fungal or bacterial count. During each of the air sampling period, bioaerosols were also collected onto polycarbonate filters of pore size of 0.4 lm with the MiniVol sampler with a PM2.5 cutoff at 5.0 l/min for the collection of endotoxins assay. Bacteria samples were collected on Trypticase Soy Agar, with cycloheximide to inhibit the growth of fungi. Agar plates of malt extract agar with chloramphenicol and rose bengal were used for the sampling and cultivation of airborne fungi (Alexopoulos and Mims 1952; Sneath et al. 1986). After sampling, bacteria plates were incubated in an inverted position at 35°C for 2 days and fungal plates at 25°C for 3–5 days. According to the standard conversion table for accounting of sterility and multiple depositions of particles at single impaction sites (Hunter et al. 1988), the transformed colony forming units (CFU) appeared to be similar to the original CFU counted. In addition, the total bioaerosols were also measured by Fluorescent Filtration Direct Count (FFDC) method developed by Moschandreas et al. (1996). Control slides were prepared by staining the pre-sterilized black membrane filter filtered with the sterilized de-ionized water loaded on the Anderson impactor, but without sampling. The slides were then observed and number of particles on the membrane filter that fluoresce in green or orange was recorded using the Leica IM50 fluorescence microscope with I3 filter at 5009 magnification (Palmgren et al. 1986). Relative humidity and temperature of the indoor/outdoor locations were recorded by using Gasprobe IAQ 4 (BW Technologies Ltd., Canada) with an interval of 1 min. 3.3 Airborne endotoxin assay Twenty-hours sampling was performed using preweighed 47-mm-diameter polycarbonate filters of pore size of 0.4 lm with the MiniVol sampler with a

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PM2.5 cut-off at 5.0 l/min. A total of two air samples per microenvironment were collected for the collection of airborne endotoxins. The maximum and minimum relative humidity and temperature for the whole sampling duration for each sampling microenvironment was measured. The sampled filters were stored in pyrogen-free 50 ml polypropylene centrifuge tube at -20°C prior to transportation to the laboratory for further treatments and were maintained at 4°C during transportation. The filters were desiccated for approximately 24 h and post-weighed to obtain the mass of samples collected. The filters were stored at -20°C prior to analysis. Blank filters were prepared as above but without the sampling. All materials coming in contact with the samples are rendered endotoxin-free by heating at 180°C for 4 h. The extraction procedure reported by Carty et al. (2003) was slightly modified with the addition of Tween 20 into the extraction water as recommended by Douwes et al. (1995). The blank or sampled filters were immersed in the pyrogen-free LAL reagent water and Tween 20 was added to a final concentration of 0.05% v/v. The immersed filters were then sonicated for 30 min at room temperature in a sonication bath with agitation at every 5 min interval. The extracts were centrifuged at 3,500 rpm for 10 min after removing the filters. Appropriate dilutions of the extracts using the pyrogen-free LAL reagent water were made prior to the LAL assay. The standard curve of absorbance versus endotoxin concentration was generated by diluting the lyophilized Escherichia coli O111:B4 endotoxin provided in the QCL-1000 LAL kit with the LAL reagent water. The standard curve ranged from 0.025 to 0.2 endotoxin units (EU). Endotoxin concentrations are reported as EU/mg PM2.5 or EU/m3 air where 10 EU is equivalent to 1 ng of reference standard endotoxin. After preparing the appropriate dilutions of the blank, sample extracts and the E. coli endotoxin standards, triplicate 25 ll aliquots of each was pipetted into a pyrogen-free 96-well, flat-bottomed polystyrene microplate. The required amount of the Chromogenic LAL and the Chromogenic substrate was loaded into the wells the same microplate well-separated from the sample aliquots. The microplate was then pre-warmed in a 37°C plate incubator for 5 min before the assay. After the incubation, 25 ll of the Chromogenic LAL was added and mixed into each well and incubated at 37°C for 15 min. 50 ll of the Chromogenic substrate was

Author's personal copy

435 160

AC bed denotes air-conditioned bedroom

Bed act denotes bedroom with cleaning activities b

a

I/O ratio Indoor/Outdoor ratio

424 339

297 149 138 11

85 0.98

1.00 0.37 435 160

424 53

42 32 191 106

127 159

64 11 85 11

64

Outdoor Office

32 0

0

21 0

Living

21

424 318 106 0.98 424 127 191 0 64 21 Kitchen

21

1,897

265 222

1,388 509

43 0.61

4.37 1,897

265 53

159 657

148 21

572 265

21 11

Bed actb

127

11

117

Bedroom

1,430

487 233

1,208 222

254 1.11

3.29 1,430

487 74

413 519

138 21

276 74

148 64

AC beda

74

42

74

Bathroom

2.1–3.3 lm 3.3–4.7 lm 4.7–7.0 lm [7 lm

Tables 2 and 3 summarize the concentration of the culturable airborne fungi and bacteria present in the microenvironments examined. In general, the bacterial counts were lower than the fungal counts in most of the microenvironments examined except for the air samples collected in the bedroom with cleaning activities. The fungal and bacterial concentrations in most of the microenvironments were fairly similar to the counts of the outdoor air samples except that they were lower in the air-conditioned office while being higher in both air-conditioned bedroom and the nonair-conditioned bedroom with cleaning activities. These variations are reflected by the indoor/outdoor (I/O) ratio. The I/O ratios (ACGIH 1989; Guo et al. 2004) refer to the relative abundance of the airborne microbes with reference to the outdoor concentration. Most ratios are close to or \1, suggesting that the mechanical or natural ventilation assisted in equilibrating indoor microbial concentrations with the outdoor microbial concentrations, or reducing them. The I/O ratios of the bacterial and fungal counts of both the air-conditioned bedroom and the non-airconditioned bedroom with the presence of cleaning activities were higher than 1 by at least a factor of 2.

Table 2 Fungal concentrations (CFU/m3) in the various microenvironments

4.1 Concentration and size distribution of airborne biocontaminants

Location

4 Results

1.1–2.1 lm

0.65–1.1 lm

Total

I/O ratio

Statistical analysis of the observational data was performed by using regression analysis. The standard deviations associated with the average values are presented in the respective column of the Table. The level of significance used in this study was P B 0.05. Spearman’s rank correlation test was performed on microbial concentrations and endotoxin level to evaluate their differences under various environmental conditions (relative humidity and temperature).

0.65–3.3 lm

3.4 Statistical analysis

3.3–7 lm

Total viable airborne fungi (CFU/m3)

subsequently added and mixed into each well and incubated for a further 6 min. 50 ll of 25% v/v glacial acetic acid was added and mixed into each well to terminate the reaction. The absorbance of each well was read at 405 nm in a microplate spectrophotometer (Gemini XS, Molecular Devices, Sunnyvale, CA).

Total

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** Bed act denotes bedroom with cleaning activities

* AC bed denotes air-conditioned bedroom

0

123

I/O ratio Indoor/Outdoor ratio

245

170 148 22 0.70 170 106 42 0 11

233

Office

11

191 54 1.00 245 21 106 64 32 11 Outdoor

11

149 117

138 95

32 0.61

0.96 233

149 21

32 95

85 11

11 0

21

95 Living

0

11 Kitchen

0

2,873 1,876 997 11.80 2,873 678 806 392 435 456 Bed act**

106

571 201

117 74

476 159 95 43

43 0.48

2.35 0.83 571 201

117 21

148 42 286 95

42 11

42 21 42 11

11

21 21 AC bed* Bedroom

32 11

21 Bathroom

11

Total 0.65–3.3 lm 3.3–7 lm

1.1–2.1 lm 2.1–3.3 lm 3.3–4.7 lm 4.7–7.0 lm [7 lm Location

Table 3 Bacterial concentrations (CFU/m3) in the various microenvironments

0.65–1.1 lm

Total

I/O ratio

Total viable airborne bacteria (CFU/m3)

Aerobiologia

The I/O ratio of the fungal count of the air-conditioned office was the lowest at 0.37 while that of the bacterial count of the bedroom with cleaning activities was the highest at 11.80. It can be clearly seen from Tables 2 and 3 that a higher proportion of the airborne culturable bacterial and fungal collected existed predominantly in the size range between 1.1 and 2.1 lm in all microenvironments. Usually, more than a third of the total culturable microorganisms sampled in most microenvironments fell into this size range of fine particulates (PM2.5). 28–66% of the culturable fungi sampled in all microenvironments were of this size range while 25–57% of the culturable bacteria were of the same size range. To better correlate the culturable and the total viable counts with the sampling of airborne endotoxin at the cut-off of 2.5 lm, the concentrations of the total viable airborne microbes estimated by the FFDC method were divided into coarse (3.3–7.0 lm) and fine fractions (0.65–3.3 lm). The finer fractions of the air samples collected appear to contain a higher microbial content, contributing to more than 50% and often close to 80% of the total viable count. The total viable bacterial counts for most of the microenvironments ranged between 117 and 2,873 CFU/m3 while those of the air-conditioned bedroom were slightly low 571 CFU/m3. The total viable bacterial counts for non-air-conditioned bedroom with cleaning activities, i.e., ‘‘bed act’’ were above 2,873 CFU/m3, about a fourfold to fivefold higher than those for most of the other microenvironments, and the total fungal concentration was also found to be very high in the same microenvironment (1,898 CFU/m3). Similarly, the concentrations of culturable bacteria and fungi were divided into the coarse and fine fractions for ease of correlation are presented in Tables 2 and 3. It can be clearly seen that more than 50% and often close to 90% of the total culturable counts were found in the fine fractions (0.65–3.3 lm) of the air samples. The size distribution of the culturable bacteria and fungi clearly shows very similar trends observed in the size distribution of the total viable count. However, the low count of the total viable microbes from the air-conditioned bedroom and the very high total viable count of the outdoor air sample did not correspond well with the level of their culturable counterparts are presented in Tables 2 and 3. This is likely to be due to the culturability of the microbes present in the air samples which was


impaired by the limited selection of culture media used. The occurrence of the Gram-negative bacteria which can contribute to the loading of the endotoxins in the air also appeared to accumulate in the finer (0.65–3.3 lm) fraction of the total bioaerosol size distribution presented in Table 5. The occurrence of Gram-negative bacteria in the larger fractions was noticed only in the air-conditioned bedroom and in the bedroom with cleaning activities. More than 50% of the culturable bacteria in the 0.65–3.3 lm range observed in the two microenvironments were Gramnegative. Less than 50% of the culturable bacteria of size 3.3–7.0 lm range from these two microenvironments were Gram-negative. For all other microenvironments, it is obvious that Gram-negative bacteria are nearly non-existent in the coarser size range of 3.3–7.0 lm. 4.2 Fungal and bacterial flora There were at least 3–18 different fungal strains cultured from each microenvironment as summarized in Table 4. The predominant fungi occurring in all locations were Aspergillus and Penicillium. There were, however, fewer and very similar numbers of bacterial strains occurring in all the microenvironments, ranging from 5 to 9 different strains. Among the indoor microenvironments, there were larger numbers of fungal strains cultured from the air of bathroom and kitchen, almost twice the number of fungal strains cultured from all other microenvironments. The bacterial strains predominantly occurring

appeared to be Staphylococcus and Micrococcus. As the bacteria were only differentiated by Gram-staining and colony morphology, the Gram-negative bacteria could not be identified without validation by biochemical tests.

4.3 Airborne endotoxin and PM2.5 level in the microenvironments Table 5 summarizes the mass of PM2.5 collected in all the microenvironments from 20 h of sampling. On average, \30 lg/m3 of PM2.5 was sampled in all microenvironments. The highest PM2.5 level of about 28 lg/m3 was collected in the kitchen while the lowest of about 12.8 lg/m3 was sampled in the air-conditioned bedroom. Except for the higher mass of PM2.5 collected from the outdoor air at 23 lg/m3 and from the kitchen, the mass of PM2.5 collected from the air of all other microenvironments ranged between 12 and 20 lg/m3. Endotoxin activities were detected in the PM collected from the 20 h sampling. Table 5 shows the endotoxin concentration in the PM2.5 samples collected from the microenvironments. On average, 300–600 EU/mg PM2.5 was found in non-air-conditioned bedroom, kitchen, living room as well as the air-conditioned office and 1,400–1,700 EU/mg PM2.5 was detected in the air of the bathroom, air-conditioned bedroom, non-air-conditioned bedroom with activities and the outdoor. The highest endotoxin level was detected in the non-air-conditioned bedroom with cleaning activities at 3,000 EU/mg PM2.5.

Table 4 Total bacterial and fungal colonies in the various microenvironments Location

Number of colonies

Identified microbes

Fungi

Bacteria

Bacteria

Fungi

Clostridium, Corynebacterium, Micrococcus, Mycobacterium, Staphylococcus, Acinetobacter, Pseudomonas, Alcaligenes and Salmonella

Acremonium, Aspergillus, Bipolaris, Cladosporium, Geotrichum, Mortierella, Penicillium, Rhizopus, Sporothrix and Trichoderma

Bathroom

18

6

AC beda

7

9

Bedroom

9

6

Bed actb

7

8

Kitchen

17

6

Living

11

6

Outdoor

17

9

3

5

Office a

AC bed denotes air-conditioned bedroom

b

Bed act denotes bedroom with cleaning activities

123


123

0 11 14 11 0.14 ± 0.1 3.338 ± 0.2 13.5 ± 0.7 599 ± 342 Office

6±4

11

0 11

11 180

138 85

42 0.20 ± 0.0

0.18 ± 0.0 1.795 ± 0.0

8.824 ± 0.4 22.9 ± 1.7

18 ± 0.4 6±2

Outdoor

39 ± 6

328 ± 127

1,717 ± 285

Living

32 11 85 21 0.20 ± 0.2 1.899 ± 0.1 28.6 ± 3.3 356 ± 280 Kitchen

10 ± 60

1,696

64 11

39 180

95 32

657 0.32 ± 0.3

0.21 ± 0.2 1.988 ± 0.03

8.236 ± 1.1 15.8 ± 2.8

15.8 ± 0.4 7±6

Bed act

37 ± 30

410 ± 344

1,720 ± 1,270

Bed room

318

11 11

42 159

64 32

53 0.33 ± 0.1

1.60 ± 1.18 1.233 ± 0.02

4.243 ± 0.01 12.8 ± 0.1

19.8 ± 0.6

1,397 ± 1,270 AC bed

18 ± 6

1,422 ± 439 Bathroom

39 ± 20

0.65–3.3 lm 3.3–7.0 lm 3.3–7.0 lm

0.65–3.3 lm

Gram-negative bacteria Gram Positive bacteria

Total count of viable bacteria

Endotoxin/microbes (EU 9 103 total microbes) Total number of microbes 9103/ lgPM2.5 PM2.5 mass concentration (lg/m3) Endotoxin (EU/m3) Endotoxin (EU/mg) Location

Table 5 Mean mass concentrations of PM2.5, endotoxin levels, mean number of microbes per mass of PM2.5, and total Gram-negative bacterial counts sampled in the microenvironments

Aerobiologia

Most guidelines for airborne endotoxin levels were expressed in terms of EU per volume of air sampled. Hence, the data were converted to the relevant metric units and are presented in Table 5 accordingly. Less than 10 EU/m3 air was measured in the air of the bedroom, kitchen, living room and the office. Close to 20 EU/m3 was detected in the air-conditioned bedroom. For most microenvironments, the endotoxin concentration in the PM2.5 collected seemed to correspond substantially well with the endotoxin concentration in the volume of air sampled except for the air-conditioned bedroom which showed a drop in the endotoxin concentration when expressed in terms of the volume of air sampled. Table 5 depicts the mean amount of microorganisms present per mass of PM2.5 sampled from the microenvironments. To obtain these data, the total viable counts (org/m3 air) gathered by the FFDC method in the 0.65–3.3 lm fraction of the air samples were divided by the mass of PM2.5 (lgPM2.5/m3) collected are presented in Table 5 to give a crude estimation of the concentration of microbes present in the airborne particulate matter. The highest level of 8,000 org/lgPM2.5 was detected in the air samples collected from the non-air-conditioned bedroom with cleaning activities and the outdoor. About 3,300–4,200 org/lgPM2.5 was found in that of the air-conditioned bedroom and the office, while the bathroom, bedroom, kitchen, and living room had the lower level at or below 2,000 org/ lgPM2.5. Table 5 presented the above depicts the amount of endotoxins produced per 1,000 microorganisms present in the air of the microenvironments. These values facilitate the estimation of relative proportion of culturable and non-culturable Gramnegative bacteria present in the total microbial concentration. To obtain these data, the concentration of the airborne endotoxins detected (EU/m3 air) as outlined in Table 5 was divided by the total viable counts (org/m3 air) gathered by the FFDC method in the 0.65–3.3 lm air fractions to get a rough estimation of the amount of endotoxins produced per 1,000 microorganisms. On average, the highest amount produced of 1.6 EU/1,000 org was detected in the air samples collected from bathroom, while a lower level of 0.14–0.33 EU/1,000 org was sampled from air samples of the other microenvironments. The values of the amount of endotoxins produced per culturable microbe were not calculated as the culturable microbes make up of \1% of the total microbial


concentration. Total microbial concentrations (bacteria and fungi) were found to be significantly correlated to endotoxin levels in all microenvironments with a correlation coefficient of r = 0.58. Similarly, bacterial and fungal concentrations were also found to be significantly correlated with each other in microenvironments with a correlation coefficient of r = 0.85.

5 Discussion 5.1 Concentration of airborne biocontaminants 5.1.1 Culturable fungi and bacteria Most of the indoor fungal and bacterial counts obtained in this study were within the local recommended limit of \500 CFU/m3 for the indoor environment except those obtained in the air-conditioned bedroom and the bedroom with cleaning activities. The fungal counts were twofold to threefold higher while the bacterial count of the bedroom with cleaning activities was almost sixfold higher than the recommended limit. Apart from the high counts in the two bedrooms mentioned above, the numbers of culturable bacteria and fungi from the other microenvironments were also comparable to the many findings reported previously on air-conditioned environments, homes, and outdoor in Singapore, Hong Kong (Guo et al. 2004) and Taiwan (Li and Kuo 1993; Su et al. 2001). Higher counts of fungi than bacteria for indoor and outdoor environments were observed. This higher count of fungi could be attributed to their ability of forming spores which survive better than vegetative cells and/or less fastidious requirements than bacteria. Bacteria can also form spores, but are limited to only several bacterial genera while most fungal species form spores. 5.1.2 Total viable count The classical method of capturing airborne microbes onto solid media as a quantitative technique for assessing aerial microbial concentrations is reportedly used in many findings (Hunter et al. 1996; Su et al. 2001). However, its severe limitations should be recognized (Griffiths and DeCosemo 1994) and have been reported often to account for \100% of total microbial populations in air samples. Karlsson and

Malmberg (1989) reported a culturable counts of onesixth of the total count while Palmgren et al. (1986) discovered that the culturable fractions could range from 1 to 100% of the total microbial counts between as well as within different environments of agricultural settings. These researchers were unable to show any strong correlation between the level of culturable bioaerosols detected and their potential for causing lung disorder. However, they showed that the total count of 109 microbes/m3 determined had been linked to acute lung impairment. The maximum total viable count detected in this study was at least 1,000-fold lower than the count obtained by Palmgren et al. (1986). The data shown in Tables 2 and 3 clearly indicates that the culturable microbes make up of \1% of the total viable. Since airborne microbes could be stressed, the number of CFU/m3 obtained is not a direct measure of viability, but only an estimation of the proportion of microbes that can be cultured on laboratory media. The limited selection of culture media used in this study clearly further heightens this fact. Relying on the growth of microbes for their detection may seriously underestimate the concentrations of cells within a sample. However, there are limited reports on total viable counts of airborne microbes in the literature probably due to the inability to identify the potential problem-causing microbes. In the outdoor, microbes are subjected to intense pressure changes caused by variable wind speeds, rapid relative humidity and temperature fluctuations, and high solar radiations. These factors can significantly decrease the culturability and/or viability of airborne microbes. This probably explains why, among all microenvironments, the outdoor air samples containing the highest total microbial count did not contain a correspondingly higher level of culturable microbes. In general, the total microbial counts for most microenvironments were several folds lower than those of the outdoor air, except for the bedroom with cleaning activities, which may be an indication of null or negligible indoor source of bioaerosols. 5.1.3 Fungus The I/O ratios of most of the fungal counts obtained were typically lower than 1 and the I/O ratio in the airconditioned office of 0.37 were comparable to the ACGIH guideline, indicating the null-existence of

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fungal contamination. The I/O ratios closer to 1 were likely to be due to the transfer of the outdoor bioaerosols into the microenvironments via natural ventilation (Hunter et al. 1996; Abt et al. 2000) that the opening of windows significantly influenced the indoor microbial concentrations. The low fungal level in the office could probably be due to the lower relative humidity of 65–73% as moisture in the air has frequently been linked to the level of aerial fungi (Brunekreef et al. 1990). However, the I/O ratio for the air-conditioned bedroom of relative humidity of 54–68% was more than 3. This could be due to the infrequent maintenance and cleaning of the filter being in use in the air-conditioning system which can act as a source of indoor bioaerosols (Guo et al. 2004), especially when the air filtration system is itself contaminated. Hence, the low I/O ratio of 0.37 in the workplace environment (office) could well be accounted for by the regular servicing and maintenance of air-conditioners. Findings reported in the literature have suggested that human activities such as walking are capable of causing an increase in the airborne concentration as a result of previously settled particles (Abt et al. 2000), and domestic activities such as cleaning and other household activities may temporarily increase the fungal concentration in the indoor air as a result of previously settled particles (Burge 1985). These factors may explain the high microbial counts in the bedroom with cleaning activities. Though moisture has been linked to the increase in indoor air fungal concentration (Meklin et al. 2003), it does not necessarily increase total viable spore concentration, but may change the composition of the airborne fungal flora (Pasanen 1992). This clearly explains that although there were 17–18 different fungal strains cultured from the air of bathroom and kitchen, almost twice the number of fungal strains cultured from all other microenvironments, the CFU/m3 counts of these two microenvironments were not higher than the rest of the microenvironments. 5.1.4 Bacteria Like the fungal counts, most bacterial counts obtained were lower than the suggested limit of 500 CFU/m3 by the NEA (1996). The I/O ratios of the bacterial counts obtained were typically lower than 1 except for airconditioned bedroom and bedroom with cleaning

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activities with a ratio of 2.35 and 11.8, respectively. The changing of bed linens, curtains and other cleaning activities appears to increase the level of bacteria in the air by up to 11-fold. Similar to the high fungal count in the bedroom with cleaning activities, the increase in airborne bacterial concentration is most probably due to the resuspension of previously settled particles (Burge 1985, 1990). This high count of bacterial and fungal contaminants resembles a transient increase in the air of the bedroom. With time, this high count is likely to drop to the level as seen in the bedroom without cleaning activities by the natural ventilation. The high counts in the air-conditioned bedroom will probably remain high if no improved ventilation and/or frequent cleaning of the air filter are exercised. Unlike fungi, relative humidity did not seem to influence the level of airborne bacteria nor changed the bacterial composition in this study. A previous study elsewhere reported (Hunter et al. 1996) that relative humidity did not have significant correlation with the level of airborne bacteria. 5.2 Size distribution of airborne biocontaminants The bioaerosols studied predominantly existed in the size range between 1.1 and 2.1 lm, usually more than 50% of the total bioaerosol collected in this study fell into this range. This trend, however, was not observed by Reponen et al. (1993) who noted that the maximum concentration of indoor fungal spores in autumn was observed in the size class of 2–3.5 lm in Finland. It is not known whether this size distribution is controlled by the characteristics of the sampling site and the local weather conditions. Interestingly, endotoxins were mostly found associated with the coarse particles in less densely populated countries such as Germany and Mexico (Heinrich et al. 2003; Osornio-Vargas et al. 2003) and with the fine particles in densely populated countries like Hong Kong (Lee et al. 2004). Unlike many other findings that reported up to a tenfold increase in the endotoxin contents in coarse particles than in fine particles, 70–80% of the ambient endotoxin level was found in the more lung-penetrating PM2.5 particles in Hong Kong. As Hong Kong is a highly populated and urbanized metropolitan city harboring largely Asians in the sub-tropical region, which, therefore, is very similar to Singapore, the case studies reported in Hong Kong may probably provide a closer reflection of what may be occurring in Singapore.


Cooking, cleaning, and the movement of people were identified as an important source of indoor particles in homes, and these sources contributed significantly both to indoor concentrations (I/O ratios varied between 2 and 33) and to altered indoor particle size distribution. Frying was found to contribute to the generation of fine and coarse particles while the combustion processes such as cooking produce fine particles and the mechanical processes such as cleaning and movement of people generate coarse particles. The slight accumulation of PM2.5 in the air samples of the kitchen above that of for outdoor air could probably be due to the fine particles generated from cooking. The null increase in PM2.5 level in the air samples of the bedroom with activities may be due to the generation of mainly coarse particles from the cleaning activities as reported by Abt et al. (2000). 5.3 Airborne endotoxin 5.3.1 Endotoxin sampling, extraction, and interlaboratory variations There are a large variety of endotoxin sampling and extraction methods available in the literature (Thorne et al. 1997; Reynolds et al. 2002; Carty et al. 2003; Heinrich et al. 2003; Long et al. 2001). Due to the relative lower abundance of airborne endotoxin in domestic situations, a greater volume of residential air is required to collect a detectable amount of aerial endotoxins than is required in occupational settings. To strike a balance between the level of noise produced by the sampler, the duration of the sampling and the ease of handling and size of sampler, the portable MiniVol was chosen for the endotoxin sampling. Polycarbonate membrane filter was chosen on top of other membrane types available due to the ease of handling and reported reproducibility of endotoxin measurements (Douwes et al. 1995; Thorne et al. 1997). Addition of 0.05% Tween 20 was included as it found to improve endotoxin extractions efficiency by about sevenfold. The first trial of endotoxin extraction performed was by sequential extractions of a sampled filter to detect further increase in endotoxin level in the subsequent extracts. The filter was first agitated in 10 ml of pyrogen-free distilled water containing 0.05% Tween 20 at 68°C for 30 min as suggested by Douwes et al. (1995) and Thorne et al. (1997). As there

was no observable dislodging of the dust from the filter, the agitation was carried out for a prolonged period of time (for over 16 h) at room temperature. The sonication of the sampled filters for endotoxin extraction (Carty et al. 2003) was incorporated after the overnight extraction procedures failed to dislodge observable dust collected on the filters and yielded negligible endotoxins. The sonication was terminated at 25 min after dislodging of dust from the filter was visually completed. The sonicated extract yielded an endotoxin level of at least threefold higher than the non-sonicated extracts. The sonication of the sampled filters was further optimized by assessing the effect of the duration of sonication on the level of endotoxin detected. It was found that a 30 min sonication yielded the highest endotoxin level. Longer sonication duration failed to improve and further reduced the endotoxin yield. The endotoxin values obtained in this study were many folds higher than the findings reported in the literature. This large variation in values has been extensively investigated by Reynolds et al. (2002), employing several groups of researchers to measure the endotoxin levels of air samples from agricultural environments. The resulting endotoxin levels measured by various laboratories spanned a wide range. The chicken dust from a chicken laying facility was found to contain 764–37,190 EU/m3, while the corn dust from an animal feed manufacturing company was found to contain 287–16,240 EU/m3. The research group (Lab D) that included the addition of Tween 20 for endotoxin extraction was consistently higher than other groups in their endotoxin values by about tenfold. Though there were significant differences in the endotoxin values, analyses from laboratories including Lab D were highly correlated. 5.3.2 Airborne endotoxin level in the indoor microenvironments The endotoxin levels detected in all the microenvironments were below 70 EU/m3, within the recommended guidelines of 100 EU/m3 (Rylander 2002). The reported mean of ambient endotoxin level was \5 EU/mgPM2.5, but the detected mean outdoor endotoxin level in this study was, many fold higher, at about 1,700 EU/mgPM2.5. Nevertheless, the endotoxin level expressed as EU/m3 reported in these findings ranged from 0.026 to 0.082 EU/m3 and was

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significantly different from that obtained in Hong Kong by Lee et al. (2004) at 94 EU/m3 which is closer to the mean outdoor endotoxin level of 39 EU/m3 obtained in this study. However, it should be pointed out that Lee et al. (2004) measured the total endotoxin level using GC–MS. Endotoxin levels detected by GC–MS are usually higher than those obtained by LAL assay. As there are no relevant reports to compare, it is not known whether the significant difference in the endotoxin level detected is partly due to the different climates and population density of places where samples were taken as discussed under Sect. 4.2. Consistent with the findings collected on the occurrence of the culturable Gram-negative bacteria and the non-culturable microorganisms (Table 5) accumulating in the fine particles, endotoxins were found to be at least fivefold higher in the air during dust-creating activities than in the non-air-conditioned bedroom and twofold higher than in the air-conditioned bedroom. As the dust-creating activities are transitional and can be quickly diluted in a wellventilated room (Abt et al. 2000), the standard deviation spanned a larger region. However, it is conceivable that such activities, when being carried out in a less-ventilated room, will remain high and pose a threat to the human health. Home humidity has been positively associated with indoor airborne endotoxin levels (Park et al. 2000), but is not entirely reflected in this study. Room airconditioning did not reduce endotoxin levels. This finding is consistent with those of Gereda et al. (2001) who found that though central air-conditioning has been associated with lower house dust endotoxin levels. Based on these observations, one can conclude that home humidity and dampness only marginally influence indoor endotoxin levels. The low endotoxin levels detected in the air samples of the air-conditioned office could be attributed to the exclusion of outdoor endotoxin levels in the closed-up office. The other microenvironments such as the non-air-conditioned bedroom and living room where both had a higher relative humidity (70–84%) than the airconditioned office (65–73%) did not exhibit higher endotoxin levels. Though both the bathroom and kitchen had a higher relative humidity (84–90%) than most of the other microenvironments, only the air samples of the bathroom exhibited a higher level of endotoxin. Similarly, a relatively higher airborne

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endotoxin level was detected in the air-conditioned bedroom of relative humidity of only 54–68%. As reported previously, humidity may not be a positive determinant factor affecting the airborne bacterial level in indoor air. It is thus plausible that relative humidity may not be a significant factor affecting the airborne endotoxin levels in the domestic settings. From Table 5, it is interesting to note that the relatively higher mass concentration of PM2.5 in the indoor air of the bathroom contained a relatively lower number of microbes, thus giving the highest value of EU/1,000 org. It is probable that the high particulate matter collected from the bathroom contained heavy chemicals such as those derived from detergent other than the bioaerosols where activities such as laundering were also carried out. It is also plausible that the total microbes captured from the bathroom air contained substantial levels of Gram-negative bacteria though they did not show up on the bacterial culture plates. Many Gram-negative bacteria are colonisers of the human gastrointestinal tract which can be released into the external environment through excretion and are frequently anaerobic microbes that cannot be cultured easily. Likewise, the low endotoxin activity from the non-air-conditioned bedroom with activity and the outdoor could be used to postulate that a large fraction of the particulate mass was contributed by other non-Gram-negative microbes or inert materials such as fibers. There was a significant correlation between total microbial concentrations and endotoxin levels, with a correlation coefficient was r = 0.58. As the microbial concentration increased, the endotoxin production level also increased as shown in Tables 2, 3 and 5. Such an association can be attributed to the presence of the endotoxin producing Gram-negative bacteria in the microenvironments. The ‘‘bed act’’ microenvironment was especially observed to have a higher level of both endotoxins and Gram-negative bacteria among the environments, confirming their strong association. There was a positive correlation between the total bacterial and fungal counts, with a correlation coefficient of r = 0.85 in all microenvironments. The bioaerosol concentrations did not show a significant correlation with temperature and relative humidity (r = 0.20 and r = 0.04, respectively). It can therefore be concluded that the microbial concentrations indoors do not really depend on environmental parameters (humidity and temperature), but they are largely related to the nature and intensity of human


activities indoors as evident from the observation made in the ‘‘bed act’’ microenvironment.

6 Conclusion In general, there were no obvious indoor bacterial or fungal sources in most of the microenvironments in the well-ventilated apartment and the air-conditioned office though there may be slight indication of higher microbial loading in the air of the air-conditioned bedroom. The airborne bacterial and fungal concentration of the well-ventilated apartment was found to be 117–2,873 and 160–1,897 CFU/m3, respectively. The predominant fungi occurring in the apartment were Aspergillus and Penicillium while the bacterial strains predominantly occurring appeared to be Staphylococcus and Micrococcus. The average indoor endotoxin level was detectable at 6–39 EU/m3. Dustcreating activities such as bed-making was demonstrated to be able to raise the aerial bacterial and fungal level by 11-fold to sevenfold, respectively, and the endotoxin level by fivefold. The degree of ventilation and the kinds of indoor activities performed may be important factors affecting the indoor level of these airborne biological contaminants. Furthermore, results of this study suggest that the amount of ventilation and the types of human activities carried out in the indoor environment are important factors affecting the level of the airborne endotoxins and airborne microbes in the microenvironments. Acknowledgments The authors thank Ms. Ong Yong Mei for her kind assistance with the collection and analysis of air samples during the field studies.

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