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Assessment of microbial contamination within working environments of different types of composting plants a

a

b

c

a

Beata Gutarowska , Justyna Skóra , Łukasz Stępień , Bogumiła Szponar , Anna Otlewska & a

Katarzyna Pielech-Przybylska a

Institute of Fermentation Technology and Microbiology, Lodz University of Technology, Łódź, Poland b

Institute of Plant Genetics, Polish Academy of Sciences, Poznań, Poland

c

Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wrocław, Poland Published online: 20 Mar 2015.

Click for updates To cite this article: Beata Gutarowska, Justyna Skóra, Łukasz Stępień, Bogumiła Szponar, Anna Otlewska & Katarzyna Pielech-Przybylska (2015) Assessment of microbial contamination within working environments of different types of composting plants, Journal of the Air & Waste Management Association, 65:4, 466-478, DOI: 10.1080/10962247.2014.960954 To link to this article: http://dx.doi.org/10.1080/10962247.2014.960954

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TECHNICAL PAPER

Assessment of microbial contamination within working environments of different types of composting plants Beata Gutarowska,1 Justyna Skóra,1,⁄ Łukasz Stępień,2 Bogumiła Szponar,3 Anna Otlewska,1 and Katarzyna Pielech-Przybylska1 Institute of Fermentation Technology and Microbiology, Lodz University of Technology, Łódź, Poland Institute of Plant Genetics, Polish Academy of Sciences, Poznań, Poland 3 Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wrocław, Poland ⁄ Please address correspondence to: Justyna Skóra, Lodz University of Technology, Institute of Fermentation Technology and Microbiology, Poland, 171/173 Wólczańska St, 90-924 Łódź, Poland; e-mail: [email protected] 1

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2

The objective of the study was to determine the degree of microbiological contamination, type of microflora, bioaerosol particle size distribution, and concentration of endotoxins in dust in different types of composting plants. In addition, this study provides a list of indicator microorganisms that pose a biological threat in composting facilities, based on their prevalence within the workplace, source of isolation, and health hazards. We undertook microbiological analysis of the air, work surfaces, and compost, and assessed the particle size distribution of bioaerosols using a six-stage Andersen sampler. Endotoxins were determined using gas chromatography–mass spectrometry (GC-MS). Microbial identification was undertaken both microscopically and using biochemical tests. The predominant bacterial and fungal species were identified using 16S rRNA and ITS1/2 analysis, respectively. The number of mesophilic microorganisms in composting plants amounted to 6.9 × 102–2.5 × 104 CFU/m3 in the air, 2.9 × 102–3.3 × 103 CFU/100 cm2 on surfaces, and 2.2 × 105–2.4 × 107 CFU/g in compost. Qualitative analysis revealed 75 microbial strains in composting plants, with filamentous fungi being the largest group of microorganisms, accounting for as many as 38 isolates. The total amount of endotoxins was 0.0062–0.0140 nmol/mg of dust. The dust fraction with aerodynamic particle diameter of 0.65–1.1 μm accounted for 28–39% of bacterial aerosols and 4–13% of fungal aerosols. We propose the following strains as indicators of harmful biological agent contamination: Bacillus cereus, Aspergillus fumigatus, Cladosporium cladosporioides, C. herbarum, Mucor hiemalis, and Rhizopus oryzae for both types of composting plants, and Bacillus pumilus, Mucor fragilis, Penicillium svalbardense, and P. crustosum for green waste composting plants. The biological hazards posed within these plants are due to the presence of potentially pathogenic microorganisms and the inhalation of respirable bioaerosol. Depending on the type of microorganism, these hazards may be aggravated or reduced after cleaning procedures. Implications: This study assessed the microbial contamination in two categories of composting plants: (1) facilities producing substrates for industrial cultivation of button mushrooms, and (2) facilities for processing biodegradable waste. Both workplaces showed potentially pathogenic microorganisms, respirable bioaerosol, and endotoxin. These results are useful to determine the procedures to control harmful biological agents, and to disinfect workplaces in composting plants.

Introduction Owing to the intense activity of microorganisms, composting plants can process organic materials into products with good fertilizing properties for agricultural use. The present study examined two types of composting plants: those processing green waste using a static pile system, and facilities producing substrates for button mushroom cultivation. The most widespread method for processing green waste is heap composting, which is undertaken either in the open air or under a cover for 6 to 8 months. During this process organic

materials (green waste from urban areas, vegetable refuse, bark, herbal waste) are formed into trapezoidal piles (windrows), and turned if the concentration of oxygen falls below 10–15% (Sharma et al., 1997). The basic raw material in plants producing substrates for button mushroom cultivation is straw mixed with horse manure, biogenic elements, and gypsum. The composting process consists of building piles, composting, turning piles, pasteurization, cooling, aeration, and inoculation with button mushroom mycelium; the process is carried out indoors, and lasts for about 3 weeks (Sharma et al., 1997).

466 Journal of the Air & Waste Management Association, 65(4):466–478, 2015. Copyright © 2014 A&WMA. ISSN: 1096-2247 print DOI: 10.1080/10962247.2014.960954 Submitted July 2, 2014; final version submitted August 26, 2014; accepted August 27, 2014.

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During composting, microorganisms, their fragments, toxins, and metabolites (MVOCs: microbial volatile organic compounds, endotoxins and mycotoxins) are released from the compost into the air, contaminating the atmosphere at the workplace and the area surrounding the facilities (Fisher et al., 2000). The predominant microorganisms in the bioaerosols of composting plants are mesophilic bacteria and fungi, although some thermotolerant microorganisms may also be found temporarily. According to European Union (EU) Directive 2000/54/EC of September 18, 2000, composting facilities are expected to exhibit some hazardous agents, which has been confirmed by many reports showing high concentrations of airborne microorganisms (104–105 CFU/m3) and the presence of potentially pathogenic microorganisms (such as Aspergillus fumigatus) belonging to group 2 biological agents (Taha et al., 2007; Nadal et al., 2009; Persoons et al., 2010). Most previous studies concern composting facilities processing green and mixed waste, while relatively few publications on composting plants producing substrates for mushroom cultivation exist (Buczyńska et al., 2008). Due to the different raw materials and dynamics of the composting process, the compositions of microorganisms in the compost and bioaerosol are also likely to differ. Studies have shown that workers of composting plants suffer from respiratory diseases more often than control groups not working at such sites (Domingo and Nadal, 2009; Nadal et al., 2009). These disorders mostly include allergies, chronic respiratory-tract diseases, and many symptoms, such as mucosal membrane irritation of the eyes and upper airways (Deportes et al., 1995; Bünger et al., 2007). The high concentrations of airborne bacteria and molds in composting facilities are thought to be the main cause of these health effects (Bünger et al., 2007). The already-mentioned Directive 2000/54/EC imposes an obligation on employers to monitor the degree of microbiological contamination of certain workplaces (According to Directive 89/391/EEC a workplace is defined as the place intended to house workstations on the premises of the undertaking and/or establishment, and any other place within the area of the undertaking and/or establishment to which the worker has access during the course of employment.) It also provides a list of 375 biological agents classified into 4 risk groups depending on the hazards they pose, and the existing prevention and treatment methods. In practice, it is impossible to monitor the presence of all these factors, and no quantitative criteria have been stipulated for acceptable level of contamination in these workplaces. Furthermore, while the methods used to date can detect only a small proportion (about 10%) of airborne microorganisms, utilizing molecular biology techniques allows the detection of microbes that do not grow on culture media under laboratory conditions. These techniques have confirmed the presence of many new microbial species in the air of composting facilities (Bru-Adan et al., 2009; Le Goff et al., 2011; Le Goff et al., 2012). However, it should be emphasized that not all microorganisms may be detected, and these laboratory methods are very expensive. Under the circumstances, it would seem useful to define indicator microorganisms or other indicator factors whose presence at a given concentration would signal a biological hazard. Such an approach has already been used for

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composting plants. Le Goff et al. (2012) and Bru-Adan et al. (2009) suggested that the total count of Saccharopolyspora and Thermoactinomycetaceae could serve as indicators for bioaerosol emission in composting facilities. Many publications have proposed that MVOC levels (Fischer et al., 2000; Tolvanen et al., 2005; Persoons et al., 2010), total counts of thermotolerant fungi and Actinobacteria (Sharma et al., 1997), or the number of gramnegative bacteria and endotoxins (Pankhurst et al., 2011) may be used as an indicator of exposure to biological agents in composting plants. Also, the total count of airborne A. fumigatus mold, which is the predominant fungal species, may be deemed a good indicator of the microbiological contamination of composting workplaces (Reinthaler et al., 1997; Domingo and Nadal, 2009). The objective of the present study was to analyze and compare the levels of microbiological contamination and the composition of microorganisms at workplaces in two types of composting plants: outdoor facilities processing green waste, and indoor facilities producing substrates for button mushroom cultivation. In addition, health risks were estimated by determining bioaerosol particle size distribution and endotoxins in dust samples, in areas with the highest degree of microbiological contamination. Finally, a new concept of assessing microbial threat in the composting plant work environment is proposed based on indicators strains.

Materials and Methods Description of the studied rooms The study involved two types of composting plants: two facilities producing substrates for industrial cultivation of button mushrooms (from raw materials such as wheat and rye straw, as well as chicken and horse manure) and two facilities processing biodegradable waste into universal organic soil conditioner (from raw materials such as green waste from urban areas, fruit and vegetable markets, and horticultural facilities). All tested composting plants were located in central Poland (flat terrain around 100–300 m above sea level). Composting plants producing button mushroom substrates were located in rural areas, and were 135 km apart (geographical coordinates are N: 51° 58ʹ 24″, E: 20° 02ʹ 55″ and N: 51° 39ʹ 34″, E: 19° 07ʹ 20″, respectively). Green waste composting plants were located in urban areas with populations of 77,000 and 710,000 people, and were 125 km apart (geographical coordinates N: 52° 16ʹ 17″, E: 18° 16ʹ 41″ and N: 51° 43ʹ 39″, E: 19° 20ʹ 52). Tests were undertaken in locations where composting was not directly conducted. Description of tested workplaces is given in Table 1. The temperature and humidity of the air in tested workplaces were measured using a PWT-401 hygrometer (Elmetron, Poland). Measurements were made from February to March 2013 (immediately prior to microbial analyses) at a height of 1.5 m from ground level, in three replicates for each location.

Analysis of microbiological contamination of air and surfaces Microbiological contamination of the air was determined using an MAS-100 Eco Air Sampler (Merck, Germany) according to

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Table 1. Characteristics of the examined workplaces in composting plants

Type of institution

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Composting plants producing button mushroom substrates

Green waste composting plants

Internal background

External background

Composting plant

Rooms

Average Cubature temperature [°C] [m3]

Average relative humidity [%]

I

Seeding hall Sell hall

7650 15000

9.4

73.1

II

Loading hall Transfer hall Sell hall

1680 12600 4900

17.8

46.6

III

Open air area

500*

21.7

30.6

IV

Waste sorting area Loading hall Machine room Offices

25317

8.4

54.9

19.1

40.9

Atmospheric air

1000 700 27–96

NT

NT

NT

Destination Samples were taken near the compost stacking machines, at the machine operator’s workplace, near the packing machine, and near the storage area for compacted compost Samples were taken near the compost stacking and control machines, during loading compost on delivery trucks, while the hall was being prepared for the next production cycle by workers cleaning surfaces Samples were taken from the waste storage area, from the site of a fresh composting pile, near a compost pile that was turned four times, from the area around composting piles, while workers were selecting composting materials and building a new composting pile Samples were taken when waste material was being sorted, when compost was being loaded into composting bins, at the process operator’s workplace— near monitoring and control devices Rooms not linked to production, situated in separate buildings and furnished with standard office equipment Samples were taken at a distance of 5–10 km from each composting plant

Note: NT - not tested

the PN-EN 13098:2007 standard. Air samples of 50 L and 100 L were collected on MEA medium (malt extract agar, Merck, Germany) with chloramphenicol (0.1%) to determine the total number of molds (including hydrophilic and xerophilic molds), and on TSA medium (tryptic soy agar, Merck, Germany) with nystatin (0.2%) to determine the total number of bacteria. Samples of air were taken at a height of 1.5 m in 3–4 representative locations of each room, at a distance of about 1.5 m from the employee. Sixteen to 18 samples were collected from each room (described later in Table 3). Samples to determine particle size distribution of fungal and bacterial bioaerosols were collected in a selected production hall of one plant producing compost for mushroom growing. Samples from work areas were collected during work and following hall cleaning (using pressure washing) in three repetitions. A six-stage Andersen sampler (model WES-710, Westech Instruments, UK) was used for size distribution analysis of bioaerosols. This instrument made it possible to divide the

bioaerosols into six fractions, in accordance with their aerodynamic diameters, as follows: ≥7.0 µm (1st stage), 7.0–4.7 µm (2nd), 4.7–3.3 µm (3rd), 3.3–2.1 µm (4th), 2.1–1.1 µm (5th), and 1.1–0.65 µm (6th). The air was sampled with a vacuum pump at a constant 28.3-L/min flow rate. Samples were collected over 5 minutes (141.5 L of air) on media: MEA medium with chloramphenicol (0.1%) (fungi) or TSA medium with nystatin (0.2%) (bacteria). Samples of air were also collected in office rooms (internal background) within each composting plant and from atmospheric air (external background) at a distance of 5–10 km from each site. Samples from surfaces (production surfaces, machinery and equipment) were collected in three repetitions using RODAC Envirocheck plates (RODAC = Replicate Organism Detection And Counting; Merck, Germany) containing either TSA medium with neutralizers (for bacteria) or Sabouraud medium (Merck, Germany) (for fungi). The traditional swab method was used for high levels of surface contamination, using saline solution (0.85%

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NaCl), swabs and metal frames (surface area 25 cm2), and the media already described. The results obtained from both methods were averaged and presented in the same units. Mature compost (after composting) from each plant was also microbiologically analyzed in three repetitions. For this purpose, samples were collected in sterile bins, and 1 g sample was weighed and mixed with 99 ml saline solution (0.85% NaCl). The samples were plated following serial dilutions from 10−4 to 10−8 on MEA medium with chloramphenicol (0.1%) (fungi) and TSA medium with nystatin (0.2%) (bacteria). All samples (air, surfaces, compost) were incubated at 30 ± 2°C for 48 h (bacteria) or at 27 ± 2°C for 5–7 days (fungi). After incubation, the colonies were counted, and the results were expressed in CFU/m3 air, CFU/100 cm2 surfaces, or CFU/g compost. The final result was calculated as the arithmetic mean of all repetitions.

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Table 2. Indicators of harmful biological agents contamination at composting plants

Microorganisms Bacteria Molds

Identification by genetic methods Bacillus pumilus Bacillus cereus Aspergillus fumigatus Cladosporium cladosporioides Cladosporium herbarum Mucor fragilis Mucor hiemalis Rhizopus oryzae Penicilium crustosum Penicilium svalbardense

Accesion numer KC182056 KF725719 KC 456184 KJ460023 KJ460022 KJ460024 KJ460025 KJ460026 KC 456186 KC 456187

Analysis of endotoxin in dust samples 3-Hydroxy fatty acids (3-OH FAs), which are unique compounds within the conserved portion of lipopolysaccharides (LPS), were used to identify endotoxins. Dust samples were collected in one location in composting plant IV (waste sorting area) and two locations (seeding hall and sell hall) in composting plant I. Samples were subjected to hydrolysis prior to 3-OH FA release, and the further steps described earlier (Sebastian and Larsson, 2003). Products of the reaction were measured by gas chromatography–mass spectrometry approach, and results were expressed in nanomoles of LPS per milligram of dust.

Identification of microorganisms All bacteria and yeasts isolates were characterized according to colony morphologies and selected biochemical tests (gram staining, catalase test, and oxidase test) (Microbiologie Bactident Oxydase, Merck, Germany). Following this, isolates were grouped into strains and identified using API tests (BioMérieux, France): API 50 CH, API STAPH, and API 20 NE. Diagnostics for yeasts were performed using the API C AUX test. Bacteria selected as indicators of microbiological contamination at workplaces in composting plants were identified using nucleotide sequences of the 16S rRNA gene according to the procedure described in the following (Jensen et al., 1993). Isolated molds were identified by macroscopic and microscopic observations following culture on CYA (czapek yeast extract agar, Difco, USA) and YES (yeast extract with supplements) media, using taxonomic keys (Klich 2002; Frisvad and Samson, 2004; Pitt and Hocking, 2009; Bensch et al., 2010). Molds and yeasts, which were specified as indicators of microbial contamination at workplaces, were identified using the ITS1/2 sequence of the rDNA region White et al., 1990. Genomic DNAs of indicator strains were extracted using the method described by Stępień et al. (2011). The resulting nucleotide sequences were analyzed and compared to those published in the National Center for Biotechnology Information (NCBI) database, using the BLASTN 2.2.27+ program (Zhang et al., 2000). The sequences obtained for microorganisms were deposited in the NCBI GenBank database (Table 2).

Selection of indicators of microbiological contamination at workplaces in composting plants Indicators of microbiological contamination were determined using adopted criteria and a scale of evaluation earlier proposed by Skóra et al. (2014), based on the frequency of strain isolation, source of isolation, and harmfulness to human health according to literature data—that is, the classification in Directive 2000/54/EC, the European Confederation of Medical Mycology (ECMM; classification BSL presented in Report of ECMM, 1996), and the Institute of Rural Health in Lublin (classification IMW presented in Dutkiewicz et al., 2007).

Mathematical calculations The arithmetic mean and standard deviation for the number of microorganisms and the frequency of species occurrence in the air and on surfaces were calculated. The frequency of species occurrence (ƒ) was calculated by dividing the number of samples in which the strain occurred by the total number of samples in each composting plant. Statistical analyses were performed using STATISTICA 6.0 software (Statsoft, USA). The results obtained for microbiological contamination of surfaces and air at the composting plants were evaluated using one-way analysis of variance (ANOVA) at 0.05 significance level. Fisher’s least significant difference (LSD) post hoc test (at 0.05 significance level) was used to verify statistical differences. Linear regression analysis was used to determine the effect of microbial contamination of the compost on microbiological contaminations of the air, in the composting plants examined.

Results The mesophilic bacterial and fungal counts in the air, surfaces, and compost at the four composting plants and control sites are given in Tables 3 and 4. In the green waste composting facilities, the total microbial count ranged from 4.2 × 103 to 3.4 × 104 CFU/m3, while in facilities producing button mushroom substrates, it ranged from 2.1 × 103 to 7.3 × 104 CFU/m3. Statistical analysis of the results of air microbial contamination did not show

M: Min: Max: SD:

IV (N = 18; n = 3)

M: Min: Max: SD: M: Min: Max: SD:

M: Min: Max: SD:

M: Min: Max: SD:

II (N = 18; n = 3)

III (N = 18; n = 3)

M: Min: Max: SD:

I (N = 16; n = 3)

M: Min: Max: SD: M: Min: Max: SD: M: Min: Max: SD: M: Min: Max: SD: M: Min: Max: SD:

104 A 103 104 104 103 A 102 103 103 102 A 101 103 103 102 A 102 103 102

8.5 6.0 3.3 1.2 5.6 1.1 1.4 4.7

2.3 5.6 6.0 2.0

1.8 2.5 4.2 1.4

× × × × × × × ×

× × × ×

× × × ×

× × × ×

103 A 101 104 104

8.6 2.5 3.1 1.2

M: Min: Max: SD:

4.6 × 102 A 0.0 9.3 × 102 4.9 × 102

9.4 1.1 2.4 8.1 1.1 1.6 4.0 1.2

1.8 7.8 7.3 7.9

1.6 1.6 3.9 1.4

6.4 9.4 3.9 1.2

1.6 8.0 4.2 1.9

× × × × × × × ×

× × × ×

× × × ×

× × × ×

× × × ×

102 A 102 103 102 103 A 102 103 103

103 A 102 103 102

104 B 103 104 104

104 AB 102 105 105

103 A 101 103 103

Composting Number of fungi in Number of bacteria plants (N; n) the air (cfu/m3) in the air (cfu/m3)

M: Min: Max: SD: M: Min: Max: SD:

M: Min: Max: SD:

M: Min: Max: SD:

M: Min: Max: SD:

M: Min: Max: SD:

1.8 2.5 5.7 2.0 1.7 3.1 5.4 1.6

4.2 2.0 8.7 2.4

3.4 4.2 8.1 2.8

7.3 9.6 4.2 1.3

2.1 9.0 5.1 2.4

× × × × × × × ×

× × × ×

× × × ×

× × × ×

× × × ×

103 A 102 103 103 103 A 102 103 103

103 A 103 103 103

104 A 103 104 104

104 A 102 105 105

103 A 101 103 103

Total number of microbes in the air (cfu/m3)

34

48

56

53

12

22

Fungi

66

52

44

47

88

78

Bacteria

Percentage contribution in the air (%)

1.5 1.7 6.2 2.3

9.0 1.6 2.3 1.2

2.4 1.8 2.9 5.2

CS

CS

M: Min: Max: SD:

M: Min: Max: SD:

M: Min: Max: SD:

M: 2.2 Min:1.6 Max:6.3 SD: 3.5

× × × ×

× × × ×

× × × ×

× × × ×

106 B 105 106 106

106 B 106 107 107

107 B 107 107 106

105 A 104 105 105

Number of microorganisms in compost (cfu/g)

Notes: N, number of air samples; n, number of compost samples; M, mean. Min/Max, minimum/maximum value; SD, standard deviation; CS, control sample; A-B, means in the same column that are followed by different letters are significantly different (Fisher’s LSD test, p < 0.05).

External background (atmospheric air) (N = 16)

Internal backbground (office rooms) (N = 16)

Green waste composting plants

Composting plants producing button mushroom substrates

Type of institution

Table 3. Quantitative analysis of microbial contamination in the air and compost in the composting plants

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Gutarowska et al. / Journal of the Air & Waste Management Association 65 (2014) 466–478 Table 4. Quantitative analysis of microbial contamination on the surfaces in the composting plants

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Type of composting plants (N)

Number of fungi on surfaces (cfu/100 cm2)

Number of bacteria on surfaces (cfu/100 cm2)

Total number of microbes on surfaces (cfu/100 cm2)

Percentage contribution on surfaces (%) Fungi

Bacteria

Plants producing button mushroom substrates (15)

M: Min Max: SD:

4.6 5.1 1.5 5.6

× × × ×

101 A 10° 102 101

M: Min: Max: SD:

1.5 1.0 6.1 2.2

× × × ×

102 A 101 102 102

M: Min: Max: SD:

3.0 1.5 6.2 2.5

× × × ×

102 A 101 102 102

15

85

Green waste composting plants (15)

M: Min: Max: SD:

1.5 1.0 2.0 5.1

× × × ×

103 B 103 103 102

M: Min: Max: SD:

1.8 7.2 3.1 1.2

× × × ×

103 B 102 103 103

M: Min: Max: SD:

3.3 2.8 4.1 6.9

× × × ×

103 B 13 13 102

46

54

Internal background (office rooms)

M: Min: Max: SD:

7.1 2.0 1.2 7.2

× × × ×

101 A 101 102 101

M: Min: Max: SD:

2.9 2.4 3.4 6.5

× × × ×

102 A 102 102 101

M: Min: Max: SD:

3.6 2.6 4.6 7.2

× × × ×

102 A 102 102 102

20

80

Notes: N, number of samples; M, mean. Min/Max, minimum/maximum value, SD, standard deviation, CS, control sample. A-B: means in the same column that are followed by different letters are significantly different (Fisher’s LSD test, p < 0.05).

significant differences across sites of the same category of composting plant and particular facilities (p > 0.05). Bacteria were the predominant group of airborne microorganisms in composting plants producing button mushrooms substrates (accounted for 78% to 88% of all microorganisms), while fungi dominated in green waste composting plants (53–56%) (Table 3). Furthermore, the air in the plant offices was also affected by composting activity, and had increased contamination compared to external air away from composting plants. Total bacterial and fungal counts in the office areas were 9.4 × 102 and 8.5 × 102 CFU/m3, respectively. Within the study period, the atmospheric air was moderately contaminated with bacteria (1.1 × 103 CFU/m3) and practically not contaminated with fungi (5.6 × 102 CFU/m3), according to the standards PN-Z-04111-02:1989 and PN-Z04111-03:1989. The facilities that showed the highest microbial counts in the compost (9.0 × 106–2.4 × 107 CFU/g) also exhibited the highest levels of microorganisms in the bioaerosol (Table 3). There were no statistically significant differences in the number of microorganisms in the compost except for one composting plant (p > 0.05). The number of microorganisms on workplace surfaces was 1.5 × 102–1.8 × 103 CFU/100 cm2 and 4.6 × 101–1.5 × 103 CFU/100 cm2 for bacteria and fungi, respectively (Table 4). The difference between the number of microorganisms on the surfaces in button mushroom compost producing plants and universal organic soil conditioner producing plants was statistically significant (p < 0.05). However, differences in the microbial contamination of surfaces between indoor composting facilities and office rooms were not statistically significant (p > 0.05). The total LPS amounts were similar in both compost facilities and ranged from 0.0068 nmol per mg of dust in composting plant IV (processing green waste) and 0.0062 nmol/mg up to 0.0140 nmol/mg in composting plant I (producing button mushroom substrates). The distribution of the fatty acids of different lengths is presented in Figure 1.

Qualitative analysis revealed a total of 75 strains isolated from the composting plants, with the largest group being molds (38 isolates), followed by bacteria (29) and yeasts (8). The largest diversity of airborne microorganisms was found in a composting plant producing button mushroom substrates, where in total of 53 strains were isolated, including 26 mold strains, 20 bacterial strains, and 7 yeast strains (Table 5). The bacteria Micrococcus lylae and Bacillus sp., were isolated at the highest frequency in both types of composting plants, while the following molds occurred at the highest frequency in these plants: Aspergillus fumigatus, Cladosporium (C. cladosporioides, C. herbarum, C. macrocarpum), and Rhizopus oryzae. The microorganisms most often isolated from the green waste composting facilities were B. pumilus, Brevibacterium sp., Geobacillus stearothermophilus, Kytococcus sedentarius, Staphylococcus epidermidis, Alternaria chartarum, Aspergillus (A. flavus, A. niger), Mucor fragilis, M. hiemalis, Penicillium svalbardense, P. crustosum, and Kloeckera sp. The species characteristic of the composting facilities producing button mushroom substrates were Geobacillus sp. (G. thermoglucosidasius), Kocuria varians, Methylobacterium mesophilicum, Micrococcus sp., Pseudomonas stutzeri, Penicillium chrysogenum, and P. spinulosum (Table 5). The following microorganisms were prevalent in the air samples collected from composting plants producing mushrooms cultivation substrates: Geobacillus thermoglucosidasius, Geobacillus sp., Methylobacterium mesophilicum, Pseudomonas stutzeri, Staphylococcus lentus, Fusarium solani, Candida krusei, and Cryptococcus laurentii. Their numbers ranged from 3.6 × 103 to 3.6 × 103 cfu/m3. The green waste composting plants predominantly had Bacillus pumilus, Geobacillus stearothermophilus, Kytococcus sedentarius, Micrococcus lylae, and Staphylococcus epidermidis (range: 1.1 × 10 3–5.8 × 103 cfu/m3; Table 5). Surfaces were

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Figure 1. 3-Hydroxy fatty acid profile in the dust from compost facilities. (a) Waste sorting area (composting plant IV). (b) Seedling hall (composting plant I). (c) Sell hall (composting plant I).

mainly colonized by Bacillus cereus, Geobacillus thermoglucosidasius, Cladosporium herbarum, and Candida guilermondii (composting plants producing substrates for mushrooms cultivation), and Bacillus pumilus, Micrococcus lylae, Staphylococcus epidermidis, S. xylosus, Mucor fragilis, Mucor hiemalis, Penicillium crustosum, and Rhizopus oryzae (green waste composting plants). Bacteria that occurred in the highest numbers (>105 colony-forming units [cfu]/g) in the composts tested were Aneurinibacillus aneurinilyticus, Bacillus licheniformis, Bacillus pumilus, Brevibacillus sp., Brevibacterium sp., and Micrococcus lylae. Based on the adopted criteria, the following 10 indicators of harmful biological agent contamination were selected: B. cereus, A. fumigatus, C. cladosporioides, C. herbarum, M. hiemalis, and R. oryzae for both types of composting plants, and B. pumilus M. fragilis, P. svalbardense, and P. crustosum for green waste composting plants (Table 5). Analysis of the distribution of bioaerosol particles in the composting plants revealed that the smallest bacterial particles (0.65–1.1 µm) constituted a significant fraction (28–38%) of all bacterial particles, while the share of such particles at the control sites amounted to 5–7% (Figure 2). Particles of sizes between 1.2 and 21 µm formed a large fraction of the bacterial aerosol in the composting premises (37–40%). They were also found to penetrate into the air of office areas (40%), while their content in the atmospheric air was small (7%). Particles with sizes between 2.2 and 11 µm accounted for 2–15% of the bioaerosol in the composting facilities, and they were predominantly present in the atmospheric air (16–27%). Bioaerosol particle size distributions were also compared between working hours and following high-pressure cleaning of the premises on completion of a production cycle. Results showed that cleaning effectively decreased the number of the smallest bacterial particles (0.65–1.1 µm) by 11% (Figure 2). A different bioaerosol particle distribution was found for fungi (Figure 2). The dominant fractions of fungal particles

present in the air of composting plants were the 1.1–2.1 µm and 2.1–3.3 µm fractions (accounting for 22–38% and 15– 42%, respectively). However, this pattern was heavily influenced by the particles present in atmospheric air (32–35%). Large fungal particles (7–11 µm) accounted for 10–16% of the fungal aerosol, in contrast to atmospheric air (6%). The cleaning of composting facilities led to a decrease in the 2.1–3.3 µm and 3.3–4.7 µm fractions. However, it also led to an increase in the concentrations of small particles (0.65–1.1 µm and 1.1–2.1 µm, Figure 3). We developed a new concept for the control of biological hazards at workplaces in composting plants, which is based on indicators of microbiological contamination. The most important stage in this method is sample selection, followed by quantitative and qualitative analysis of microbial contamination in the selected workplaces (Figure 4). Contamination should controlled at places likely to have biological agents. In order to select the right workplaces for analyses the following questions must be asked: What activities are performed at the workplace? Are bioaerosols potentially produced during such activities? Is there is a high concentration of biological factors during work? What is the frequency of those activities? What is the exposure time to biological factors? How many employees work at each workplace? Are any employees at higher risk from such factors, for example, pregnant women? Is there is an injury risk? Were any accidents or hazardous incidents to workers’ health reported? One should consider controlling biological hazards in composting plants within production equipment operator workplaces, those with high dust concentrations and where personnel come in direct contact with organic waste. In the next step, the microbial contamination of designated places should be analyzed. This should include examining the total number of bacteria and fungi in the air, and the endotoxin content of the air/dust. In the case of air analysis, at least 3 replicates must be taken from a height of approximately 1.5 m (height of an employee’s mouth and nose)

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Microorganism Bacteria Aneurinibacillus aneurinilyticus Bacillus cereus Bacillus lentus Bacillus licheniformis Bacillus megaterium Bacillus pumilus Bacillus sp. Bacillus subtilis Brevibacillus sp. Brevibacterium sp. Brevundimonas vesicularis Erysipelothix rhusiopathiae Geobacillus sp. Geobacillus stearothermophilus Geobacillus thermoglucosidasius Kocuria rosea Kocuria varians Kytococcus sedentarius Leuconostoc mesenteroides ssp. Methylobacterium mesophilicum Micrococcus lylae Micrococcus sp. Propionibacterium avidum Pseudomonas stutzeri Sphingomona paucimobilis Staphylococcus epidermidis Staphylococcus lentus Staphylococcus sciuri Staphylococcus xylosus Molds Alternaria chartarum Aspergillus flavus Aspergillus fumigatus Aspergillus niger Aspergillus ochraceus Aspergillus parasiticus Botrytis cinerea Chrysosporum pannarum Cladosporium cladosporioides Cladosporium herbarum Cladosporium macrocarpum Cladosporium sphaerospermum Eurotium amstelodami Fusarium culmorum Fusarium solani Fusarium sp. Fusarium sporotrichoides Geotrichum candidum Mucor fragilis Mucor hiemalis Pencillium implicatum Penicillium svalbardense Penicillium chrysogenum Penicillium commune Penicillium coprobium

BM 0/0/0 56/50/0 40/0/0 0/0/38 0/0/0 0/0/0 80/0/63 0/0/0 0/0/63 0/0/0 0/0/38 25/0/0 89/0/0 0/0/0 61/83/0 0/0/0 80/0/0 0/0/0 56/17/0 78/0/0 90/0/100 67/0/0 0/0/0 78/0/0 0/0/0 0/0/0 100/0/0 30/0/0 0/0/0 0/0/0 0/0/0 33/0/0 0/0/0 0/0/0 17/0/0 6/0/0 10/0/0 100/17/0 56/33/0 50/0/0 30/0/0 6/0/0 17/0/0 33/0/0 0/0/0 0/0/0 0/0/0 0/0/0 10/0/0 40/0/0 0/0/0 83/0/0 10/0/0 40/0/0 0/0/0 22/0/0

GW

Presence in the outdoor air

40/0/57 40/0/0 0/0/0 0/0/66 50/0/0 86/100/75 30/0/36 40/0/0 0/0/61 80/0/75 0/0/50 0/0/0 0/0/0 100/0/0 0/0/0 30/0/0 0/0/0 90/0/0 0/0/0 0/0/0 100/100/0 0/0/0 42/0/0 0/0/0 0/0/67 100/100/0 0/0/0 0/0/0 70/25/0 64/0/0 64/0/0 79/0/14 79/0/0 10/0/14 0/0/0 0/0/0 0/0/0 79/0/0 30/0/0 86/0/4 0/0/0 0/0/0 0/0/0 0/0/0 0/0/11 14/0/0 0/0/18 71/25/29 50/25/0 0/0/0 90/0/0 0/0/0 0/0/0 80/0/0 100/25/0 0/0/0

– – – – – + + + – – – – – – – + + + – – + + – – + + + + + + + – + + – + – + + – – – – + + + – – – – – + + – + –

Number of microorganisms Air/Surfaces/Compost (cfu/m3; cfu/100 cm2; cfu/g) BM 0/0/0 2.9 × 102/2.8 × 101/0 1.3 × 101/0/0 0/0/3.4 × 105 0/0/0 0/0/0 2.8 × 102/0/5.3 × 103 0/0/0 0/0/1.2 × 105 0/0/0 0/0/1.7 × 104 3.0 × 101/0/0 3.6 × 103/0/0 0/0/0 2.9 × 103/6.1 × 101/0 0/0/0 1.1 × 102/0/0 0/0/0 3.8 × 102/1.7 × 10°/0 2.8 × 103/0/0 4.4 × 102/0/8.4 × 106 1.3 × 101/0/0 0/0/0 3.5 × 103/0/0 0/0/0 0/0/0 2.8 × 103/0/0 125,4/0/0 0/0/0 0/0/0 0/0/0 2.2 × 101/0/0 0/0/0 0/0/0 2.2 × 10°/0/0 4.3 × 10°/0/0 3.0 × 10°/0/0 8.6 × 10°/3.8 × 10°/0 1.5 × 102/1.9 × 101/0 8.6 × 10°/0/0 3.6 × 101/0/0 3.0 × 10°/0/0 1.8 × 102/0/0 1.9 × 103/0/0 0/0/0 0/0/0 0/0/0 0/0/0 3.1 × 101/0/0

GW 7.4 × 101/0/1.7 × 105 2.0 × 101/0/0 0/0/0 0/0/1.6 × 106 0/0/0 1.1 × 103/4.3 × 102/1.3 × 106 2.0 × 101/0/1.5 × 104 1.3 × 101/0/0 0/0/2.0 × 105 6.1 × 101/0/2.3 × 105 0/0/1.4 × 105 0/0/0 0/0/0 4.2 × 103/0/0 0/0/0 1.8 × 101/0/0 0/0/0 5.8 × 103/0/0 0/0/0 0/0/0 4.2 × 103/5.8 × 101/0 0/0/0 8.0 × 101/0/0 0/0/0 0/0/2.3 × 105 4.4 × 103/1.7 × 102/0 0/0/0 0/0/0 4.3 × 101/2.2 × 101/0 1.0 × 102/0/0 5.1 × 101/0/0 1.7 × 10°/0/1.8 × 102 5.0 × 102/0/0 2.1 × 101/0/1.0 × 101 0/0/0 0/0/0 0/0/0 1.6 × 102/0/0 3.0 × 101/0/0 1.4 × 102/0/1.3 × 101 0/0/0 0/0/0 0/0/0 0/0/0 0/0/5.9 × 101 3.4 × 10°/0/0 0/0/2.1 × 102 1.8 × 102/3.2 × 102/6.7 × 102 4.4 × 101/3.2 × 102/0 0/0/0 6.0 × 102/0/0 0/0/0 0/0/0 9.8 × 102/0/0 7.7 × 102/3.0 × 101/0 0/0/0

(Continued )

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Table 5. (Cont.) Frequency of isolation in all samples in composting plants (%), air/surfaces/compost

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Microorganism Penicillium crustosum Penicillium expansum Penicillium glabrum Penicillium griseofulvum Penicillium italicum Penicillium janczewskii Penicillium lividum Penicillium spinulosum Phoma sp. Rhizopus oryzae Scopulariopsis brevicaulis Scopulariopsis candida Trichoderma koningii Yeast Candida albicans Candida guilermondii Candida krusei Cryptococcus laurentii Kloeckera spp. Rhodotorula glutinis Rhodotorula minuta Rhodotorula mucilaginosa

BM 40/0/0 0/0/0 6/0/0 0/0/0 22/17/0 83/0/0 0/17/0 17/33/0 33/0/0 0/0/0 50/0/0 20/0/0 0/33/0 33/0/0 17/0/0 0/0/0 33/0/0 0/17/0 20/0/0

GW

Presence in the outdoor air

0/0/0 0/0/11 0/0/0 0/0/14 0/0/0 0/0/0 0/0/0 0/50/0 0/0/0 0/0/14 0/0/4 0/0/0 0/0/0 0/0/0 0/0/0 86/0/0 0/0/0 20/0/4 0/0/0

+ – – – – + – + – – + – – – + – + – +

Number of microorganisms Air/Surfaces/Compost (cfu/m3; cfu/100 cm2; cfu/g) BM 1

1.6 × 10 /0/0 0/0/0 3.3 × 10°/0/0 1.6 × 101/0/0 1.8 × 102/0/0 0/0/0 8.6 × 10°/0/0 2.5 × 101/0/0 0/0/0 1.3 × 101/0/0 0/0/0 2.2 × 10°/2.8 × 10°/0 2.2 × 101/0/0 0/4.7 × 10°/0 7.7 × 101/1.9 × 10°/0 9.0 × 101/0/0 0/0/0 1.7 × 101/0/0 6.3 × 10°/0/0 0/1.3 × 101/0 1.7 × 103/0/0 1.3 × 103/0/0 0/0/0 6.0 × 101/0/0 0/4.7 × 10°/0 9.2 × 10°/0/0

GW 0/0/0 0/0/5.9 × 101 0/0/0 0/0/6.8 × 101 0/0/0 0/0/0 0/0/0 0/8.1 × 102/0 0/0/0 0/0/4.4 × 101 0/0/5.7 × 10° 0/0/0 0/0/0 0/0/0 0/0/0 7.5 × 101/0/0 0/0/0 4.6 × 10°/0/5.9 × 102 0/0/0

Notes: BM, composting plants producing button mushroom substrates, GW, green waste composting plants; boldfaced microorganisms are proposed as indicators of contamination of harmful biological agents in working environment in composting plants.

Figure 2. The contribution of bacterial bioaerosol at workplaces in composting plant I.

Figure 3. The contribution of fungal bioaerosol at workplaces in composting plant I.

The number of repetitions and sampling locations should be correlated with either the cubature of the room, or the area occupied by the employee while performing his or her duties. Methodological details for the study of bioaerosols in the workplace can be found in the standards: EN 13098:2000; EN 14031:2003; EN 14042:2003; and EN 14583:2004.

Bacterial endotoxin can be tested from air samples according to the standards (Limulus assay), or from dust using gas chromatography–mass spectrometry (GC-MS) as presented in the present study. The results of quantitative analysis should be based on the limits set by individual countries, due to a lack of panEuropean standards (according to the Polish Committee for

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Figure 4. Guidelines for the control of microbial hazards in composting plants.

the Highest Permissible Concentrations and Intensities of Noxious Agents in the Workplace: for mesophilic bacteria 1 × 105 CFU/m3; for fungi 5 × 104 CFU/m3; for endotoxin 200 ng/m3 to 2000 EU/m3). The next stage of control is verifying indicator strains that are representative of harmful biological agent contamination in composting plants (Table 2). Verification can be carried out either by cultivation or by using a genetic method. The nucleotide sequences of indicator strains have been deposited in the NCBI GenBank database (Table 2). The absence of indicator microorganisms implies a high probability for the lack of biological hazards within the workplace. It is recommended that such workplaces follow basic hygiene rules. Detecting indicator species during microbial analysis of composting facilities provides a warning for possible health hazards; hence, appropriate safety measures must be taken. These include wearing personal protection (filtration half masks, gloves), and following preventive measures such as disinfection and dust reduction.

Discussion A comparison of microbial contamination between the two types of composting plants showed that the degree of air contamination does not depend on the type of composting facility (no statistically significant differences, p > 0.05), but is specific to a given plant. The number of microorganisms in the processed compost primarily influences the degree of air contamination in composting facilities (determination coefficient R2 = 0.9906;

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correlation coefficient r = 0.9858). An additional source of airborne microorganisms is atmospheric air. The total airborne bacterial and fungal counts in the workplaces were 1.6 × 103–6.4 × 104 CFU/m3 and 4.6 × 102–1.8 × 104 CFU/m3, respectively. Total microorganisms count in composting plants did not exceed the quantitative reference thresholds specified by the Polish Committee for the Highest Permissible Concentrations and Intensities of Noxious Agents in the Workplace (Skowroń and Górny, 2012), which is 1.0 × 105 CFU/m3 for bacteria and 5.0 × 104 CFU/m3 for fungi count. Contamination in all other plants did not exceed the permissible values. The results of quantitative measurements are similar to those obtained for composting plants by other authors. The previously reported total bacterial and fungal counts for green waste composting plants were 2.0 × 103–2.0 × 105 CFU/m3 and 2.8 × 102–1.0 × 105 CFU/m3, respectively (Taha et al., 2007; Nadal et al., 2009; Persoons et al., 2010). The corresponding values for a composting facility producing button mushroom substrates were 8.6 × 103 CFU/m3 and 6.4 × 104 CFU/m3, respectively (Buczyńska et al., 2008). The number of microorganisms in composting plant workplaces largely depends on the type of processing. According to Taha et al. (2007), in static compost processing, it amounts to 103–104 CFU/m3, while in active compost processing, this figure is higher by 1 log unit. The values reported in the present work are for mature compost (not mixed or turned), which may be the reason for lower levels of air contamination. The microbial concentrations in samples of compost (2.2 × 104–2.4 × 107 CFU/g) were similar to the results reported by other authors (Sharma et al., 1997). We observed moderate amounts of similar endotoxin markers in the settled dust, identified by 3-OH FAs, in both compost facilities. Positive correlation of biological activity of LPS has been previously related to the presence of shorter fatty acids in lipid A (3-OHC10:0, 3-OHC12:0, 3-OHC14:0), while longer and odd-numbered acids were not correlated with endotoxin activity (Park et al., 2004). In the present study, biohazard indicators included two species of the genus Bacillus (B. cereus and B. pumilus). Bacillus cereus, selected for both types of composting plants, occurred mainly in the air in high concentrations. On the other hand, B. pumilus was present in green waste composting plants in the air, on surfaces, and in the compost. Bacillus cereus may either exhibit toxicity in workers exposed to it, as a result of ingestion or contact with airborne dust or droplets (through enterotoxins causing food poisoning), or give rise to an infectious disease (pneumonia) (McKillip, 2000). Bacillus pumilus is also considered to be an etiological factor of food poisoning, and can cause skin infections (Kayser et al., 2005). According to the literature, the most common sources of Bacillus include soil, dust, plants, and, in the case of B. cereus, also food products (McKillip, 2000; Kayser et al., 2005). According to Dutkiewicz et al. (2007), B. cereus and B. pumilus mostly affect occupational groups such as farmers engaged in mixed (animal and plant) production, warehouse workers dealing with raw materials and products of plant and animal origin, and workers in the grain industry. Thus, these bacteria could be indicators for hazardous biological agent

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contamination in composting facilities; they enter these facilities via the raw materials, and spread due to the favorable conditions created by the composting processes. Among the fungi isolated from the composting plants, the following molds were found to be indicators of contamination with harmful biological agents: Aspergillus (A. fumigatus), Cladosporium (C. cladosporioides, C. herbarum), Mucor (M. fragilis, M. hiemalis), Penicillium (P. crustosum, P. svalbardense), and Rhizopus (R. oryzae). Aspergillus fumigatus has been isolated from compost and air in many composting facilities to date. A number of authors have reported that it is harmful to human health due to its allergic, toxic, and invasive effects on composting facility workers (Syzdek et al., 1995; Fisher et al., 1999). Furthermore, allergens of the mold A. fumigatus, as well as those of C. cladosporioides and C. herbarum indicator species, have been characterized in terms of structure and biological function by the World Health Organization and the Allergen Nomenclature Sub-Committee of the International Union of Immunological Societies (International Union of Immunological Societies Allergen Nomenclature Subcommittee, 2013). Dutkiewicz et al. (2007) stated that molds of the Penicillium genus constitute a work hazard that may cause exposed workers to suffer from alveolitis allergica, bronchial asthma, allergic rhinitis, and allergy-related conditions. These molds are known to produce citrinin, citreoviridin, cyclopiazonic acid, secalonic acid D, patulin, rubratoxin A and B, and viridicatin (Pitt and Hocking, 2009). The Penicillium species, proposed as indicators of contamination with noxious biological agents in composting plants, were subjected to laboratory tests and found to produce enniatins (P. svalbardense) and hydrolyzed fumonisin B1 (P. crustosum). In turn, Mucor and Rhizopus indicator species may lead to zygomycosis of the lungs, central nervous system, gastrointestinal tract, and other organs (Kulkarni et al., 2011). All the mold species proposed as indicators of contamination with noxious biological agents are characteristic of the composting environment. They are resistant to the high temperatures of composting processes (similarly to Bacillus spores), and their presence at such workplaces has been reported in previous studies. A comparison of the aerodynamic size distributions of bioaerosol particles showed differences between bacterial and fungal populations. Given the aerodynamic particle distribution of the bacterial aerosol and the cell sizes of the airborne bacteria present in the composting facilities, one can conclude that the predominant particle fractions (0.65–1.1 µm and 1.2– 2.1 µm) may comprise of individual bacterial cells. Such small particles penetrate the bronchial tree the most deeply, and reach pulmonary alveoli, posing the greatest health hazard (Kulkarni et al., 2011). Larger particles (2.2–11 µm), which may reach the upper regions of the respiratory tract (the nasal cavity, pharynx, trachea, and bronchi), mostly came from atmospheric air and contained cell aggregates: two- or three-phase systems on solid or liquid carriers, which are typical of bioaerosols. A comparison of bioaerosol particle distributions in the air of composting facilities during work and following pressure cleaning showed that the number of the smallest particles in the bacterial aerosol declined by 11%. In contrast, the number

of larger aggregates increased, as bacterial cells probably stuck together, forming clusters on the surface of water droplets. Thus, cleaning efficiently reduced the most severe bacteriarelated inhalation hazard. The pattern exhibited by fungal bioaerosol particles was different from that of the bacterial aerosol. The predominant particles were larger (1.1–2.1 µm, 2.1–3.3 µm, and 7–11 µm agglomerates). The main fungal components corresponding to these sizes of aerodynamic particles are spores, mycelial fragments, and agglomerates of mycelium with spores. In the case of fungi, the cleaning process led to increased numbers of the smallest particles (0.65–1.1 µm and 1.1–2.1 µm), which may be attributable to the breakup of airborne agglomerates into individual spores and isolated mycelial fragments, as well as to the activation of fungal spores as a result hydration. Thus, the process of pressure cleaning was found to increase the risk of inhalation of fungal species. A previous study by Byeon et al. (2008) showed that the smallest aerodynamic particles (0.65–1.1 µm) present in the bioaerosol in an outdoor composting plant posed a considerable health hazard. Moreover, based on the particle distribution changes of the microorganisms present in the bioaerosols over composting time, they reported that the greatest risk occurred at the beginning of the composting process. The present study confirmed that composting plants producing button mushroom substrates pose a risk of inhalation of small bacterial and fungal bioaerosol particles. At the same time, this hazard may be reduced or aggravated after a cleaning procedure, depending on the type of microorganisms. The risk to workers’ health in both types of composting plants (producing button mushroom substrates and processing green waste) may also be due to potential pathogens and endotoxins whose presence was proven in the present study. We thus recommend the development of guidelines for the control of harmful biological agents, and methods for removing biological threats for these types of plants. The guidelines developed for assessing microbiological hazards at workplaces is advantageous, since they use available and inexpensive methods, and follow clear handling procedures. The currently applicable European standards (EN 13098: 2000, EN 14031: 2003, EN 14042: 2003, EN 14583: 2004), which should be used in the analysis of composting plant workplaces, specify the methods to test microorganisms and bacterial endotoxins in the air. The quantitative assessment of endotoxin in composting plants is difficult task due to presence of disturbing compounds in these environments. Therefore, according to the authors, it is preferable to use an instrumental method, with detection of endotoxin markers from settled dust samples. The threshold values for chemical markers of endotoxin have not been officially stated, but results obtained by this approach at different workplaces were published in several papers (Paba et al., 2013). Globally, no legal limits are set for the number of microorganisms in work environments. In 2012, the Polish Committee for the Highest Permissible Concentrations and intensities of Noxious Agents in the Workplace published proposals for such limits (Skowroń and Upper 2012), which the authors have have adopted for this work.

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The directive on the protection of workers from risks related to exposure to biological agents at work (Directive 2000/54/ EC) provides a list of almost 400 harmful biological agents. However, testing the presence of all of these at the workplace is a difficult, time-consuming, and expensive task. We describe a new concept where detecting indicator microorganisms would signal the presence of biological hazards. Furthermore, their presence can be detected by cultivation methods or rapid genetic techniques. Particle size distribution can be useful in cases where workers have health problems, which may be caused by biological factors. The volumetric sampling method for this analysis is consistent with the already-mentioned standards, and the device used for this purpose is a six-stage Andersen sampler (Byeon et al., 2008). Occupational medicine physicians can use particle size distribution for occupational disease jurisprudence. The study of the relationship between the concentration of biomolecules on various aerodynamic diameters and associated health effects is a key element to establish legally binding limits for microbial contamination at the workplace. Such studies should be done in the future. Additionally, we recommend both the setting of limits for endotoxin concentration, measured by GC-MS, in dust and developing guidelines for removing biological threats, for composting plants.

Funding Studies were conducted within a project of the Polish National Center for Research and Development coordinated by the Central Institute for Labour Protection–National Research Institute, number III.B.03, “Development of principles for evaluation and prevention of hazards caused by biological agents in the working environment using indicators of microbial contamination.”

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About the Authors Beata Gutarowska is professor LUT and the director of the Institute of Fermentation Technology and Microbiology, Lodz University of Technology, Łódź, Poland. Justyna Skóra is a Ph.D. student and Anna Otlewska and Katarzyna Pielech-Przybylska are assistant professors at the Institute of Fermentation Technology and Microbiology, Lodz University of Technology, Łódź, Poland. Łukasz Stępień is the deputy head of the Department of Pathogen Genetics and Plant Resistance and Leader of Plant-Microorganism Interaction Team at the Institute of Plant Genetics, Polish Academy of Sciences, Poznań, Poland Bogumiła Szponar is a researcher at the Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wrocław, Poland.

Assessment of microbial contamination within working environments of different types of composting plants.

The objective of the study was to determine the degree of microbiological contamination, type of microflora, bioaerosol particle size distribution, an...
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