Bioaerosol emitted in university biological laboratories may exert adversely effects on employees and students. The occupational health and safety have been given much importance. This study aimed to monitor the risk of bioaerosol formation inside a biological wastewater treatment laboratory where other biological activities were performed along with wastewater treatment using the biological method. A single-stage impactor performed the air sampling to study concentration of bacterial and fungal bioaerosols and the diversity of air microbiota also been assessed. In early winter, 2018 MPN/m3 bacterial aerosol concentrations were observed which correlated with an increased number of occupants and their experimental activity. In contrast, the fungal concentration was found below the upper permissible limits in indoor environments, and it was influenced by seasonal change and humidity and were not influenced by number of occupants. The more significant number of morphological types of bacteria and fungi indicated diversity of air microbial community. This result suggests a moderate risk of bacterial bioaerosol to occupants working in wastewater treatment laboratory.
Short Report
Challenges and opportunities: A case study from a European city shows the influence of biological work on bioaerosol formation from indoor biological wastewater treatment laboratory
https://doi.org/10.21203/rs.3.rs-2849603/v1
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Indoor air
Biology Laboratory
Bioaerosol
Air microflora
Indoor air has been given significant importance for improvisation in occupational health and safety. The characterization of indoor air pollutants such as Particulate Matter 2.5, Particulate Matter 10, CO2 concentration, airborne particles etc. is required to assess the quality of respirable air and give an idea about the health risk associated with polluted air. Bioaerosols are airborne particles, very small in size ranging from 0.001 to 100 µm. It comprises of both living and non-living entity such as bacteria, fungi, virus, pollen and also metabolic products such as endotoxin or mycotoxin (Michałkiewicz et al., 2018). When bioaerosols get entered inside the respiratory tracks and deep into lungs, it prompts several adverse health effects, including allergies, pneumonia, rhinitis, asthma, tuberculosis, bronchitis and cardiovascular diseases (Breza-Boruta 2016; Douglas et al. 2017).
Indoor air quality and degree of bioaerosol generation depends on various factors like spatial temporal variation, seasonal variation, air relative humidity (Rh%), temperature, ventilation etc (Alves et al. 2016; Balasubramanian et al. 2012; Knudsen et al. 2017). Indoor environment is more complicated in structure. Total concentration and biodiversity of bioaerosol present in indoor laboratory environments like university research laboratory (Singh et al., 2022) clinical biochemistry laboratory (Dubuis et al., 2021), fish toxicity laboratory ( Hwang et al., 2017), animal laboratory (Hwang et al., 2017), wastewater treatment plant (Han, Li, et al., 2020) has been reported. Its condition relates to the processes going on, people working there, and condition which includes cleanliness of surfaces or materials (Adams et al. 2013; Heo et al. 2017). In laboratories, especially those specializing in biological work, workers have a relatively high chance of being exposed to bioaerosols. It may be introduced from outdoors or a contaminated ventilation system, and from handling biological samples (Hwang et al. 2013, 2017). In addition to creating adverse health outcomes, these agents can contaminate work performed in the laboratory.
The gallons of wastewater with various types of pollutants are treated at wastewater treatment plants (WWTPs). According to published research (Singh et al., 2022; Wolany & Płaza, 2017), the bioaerosol emission from WWTP is influenced by the process stage and the method employed to aerate and mix the sewage in bioreactors. The problem of wastewater treatment affects the entire world, and numerous researchers are working to find new or improved pollutant removal or degradation technologies. The breakdown of xenobiotic pollutants such synthetic dyes, pharmaceutical active compounds (PhACs), polychlorinated biphenyls (PCBs), and other pollutants has drawn attention to bioremediation technology during the past 10 years and made it into the green technology category (Jureczko et al., 2021; Upadhyay & Przysta, 2022). To the best of our knowledge, however, there hasn't been much research done on the dangers and likelihood of bioaerosol generation in biological wastewater treatment labs. There is a high demand to provide regulation of air quality in Poland as not very specified norms are available (Wolny-ko & Malinowski, 2017). Hence, it emphasized the importance of studying bioaerosol at laboratories, gathering data and making standards for acceptable concentrations of bioaerosol, and implementing solutions that will eliminate potential threats to people working in laboratories.
To address the identified research gap, it is necessary to formulate the bioaerosol analysis in a university laboratory where experiments for remediation of different pollutants is performed via biological treatment. The main purpose of this research is to measure the total concentration of mesophilic bacteria, psychrophilic bacteria and culturable fungi and observe the biodiversity of microflora with respect to seasonal variation, temperature and humidity. The secondary objective is to check the effect of other parameters on the concentration of bioaerosols, such as biological work carried out in the laboratory, number of occupants, cleanliness, and ventilation etc. Overall, the findings of this study will reiterate the concern of biological air pollution in research laboratories and will represent an inevitable aspect for the validation of bioaerosol exposure in laboratory-scale biological wastewater treatment system.
2.1 Sampling Site Characterization
The biological wastewater treatment laboratory was selected for bioaerosol emission monitoring. The laboratory is located in Silesian University of Technology premises, on the second floor of building namely Centre of New Technology in the middle of the town of Gliwice, Upper Silesia, Poland (50.293865847617234, 18.682216489638957) (Fig. 1). Total five locations were chosen for sampling and its characteristics is provided in Table 1. The air sampler was placed on the working table in laboratory where different activities like preparation of culture media, phytotoxicity test, zoo toxicity test, treatment of wastewater containing synthetic dye or cytostatic drugs with help of fungi or bacteria species etc. are carried out. Air sample was collected from the height of 1 m above ground level. The sampling was performed once in a week for three months. The three different time-period was chosen for study to check effect of seasonal variation: summer (23 May, 2022 to 15 June, 2022), Autumn (19 September,2022 to 10 October, 2022) and Early winter (14 November,2022 to 5 December,2022). In university, July to September is a holiday, so laboratory work slows down during this period.
| Location | Lab Facilities | Type of biological work or other | Ventilation, UV cleaners, cleanliness | Area (m2) | Number of People working |
|---|---|---|---|---|---|
| 229a | Laminar Air Flow Cabinet, Hygroscopic chamber, Incubator (26 ℃), Instruments related to Air monitoring | Not related to inoculation of microorganisms, pouring plates, washing area | Good, No, Good | 29.66 | 4 ± 4 |
| 229b | Biosafety Cabinet, Shaker, Colony Counter, Refrigerator | Inoculation of bacteria, fungi, actinomycetes, experiment related to bioremediation of wastewater containing synthetic dye, cytostatic drug | Good, Yes, Good | 24.05 | 2 ± 3 |
| 229c | Incubator (37 ℃, 20 ℃), Autoclave, Water bath, Refrigerator | Media Preparation room, content for decontamination complied for many days | Good, No, Not fairly good | 12.09 | 2 ± 1 |
| 231a | Plant growth chamber, centrifuge, microscope, reactor, spectrophotometer | Daphnia magna and Lemna minor grow for toxicity test, measurement of dye degradation in fungal sample | Not fairly good, Yes, Not fairly good | 23.12 | 2 ± 2 |
| 231 | Corridor near to lab | No microbiological activity, passage of air from lab 231a via door | Intermediate, No, Good | 7.28 | - |
2.2 Bioaerosol Sampling and Observation Method
Bioaerosols sampling was carried out using BIOMERIEUX Air IDEAL 3P single stage impactor. The filters of the air sampler were sterilized by autoclaving before sampling and the surface of sampler was sterilized between each measurement with 70% ethanol. The volume of air samples was adjusted to 100 L (0.1 m3), or 200 L (0.2 m3). According to protocol used by Brągoszewska et.al. (Brągoszewska, 2019), the air samples were impacted onto petri plates of 90 mm diameter containing two different culture mediums: Tryptic Soy Agar (TSA- Pancreatic digest of casein – 15 g/L, Soy peptone – 5 g/L, Sodium chloride – 5 g/L, Agar – 15 g/L) or Sabouraud Dextrose Agar (SDA- Yeast extract – 2 g/L, Peptone – 3 g/L, Peptone SP – 3 g/L, Peptone K – 3 g/L, Glucose – 19 g/L, Dipotassium hydrogen phosphate – 0.5 g/L, Potassium dihydrogen phosphate − 0.5 g/L, Chloramphenicol – 0.5 g/L, Agar – 13 g/L ) with Chloramphenicol. To determine the total number of air-borne mesophilic bacteria (M) and psychrophilic bacteria (P), Tryptic Soy Agar (TSA) medium was used for growth of microorganism and petri plates were incubated at 37 ± 1 ℃ and 20 ± 1 ℃ respectively. Mesophilic bacteria were observed after 48 h incubation whereas psychrophiles were observed after 96 h of incubation. TSA allowed growth of both bacteria and fungi as no antibiotic was added. For selective growth of fungi (F), Sabouraud Dextrose Agar (SDA) with Chloramphenicol was utilized. This medium intended for cultivation of pathogenic and commensal fungi and yeast, with chloramphenicol added to inhibit bacterial growth. SDA plates were incubated at 25 ± 1 ℃ for 6 days. All sampling were taken in triplicates. The relative humidity (RH%) and temperature (℃) has been recorded each time to check the influence of humidity and temperature on the diversity of aerosols and find effect on number of mesophilic, psychrophilic and fungi. Following the incubation time, the morphology of bacterial or fungal colonies were observed for pigmented and non- pigmented bacterial colonies and colony characteristics. The total number of colonies were enumerated using colony counter and airborne concentrations of bacterial and fungal aerosols, in colony-forming units per cubic metre (CFU/m3), were calculated for each variation (Eq. 1). It is important to note that although direct measurement of the concentration of living airborne microorganism is extremely difficult, hence, the CFU/m3 is commonly used substitute of the concentration of living microorganisms present in the air. The CFU was corrected and considered as most probable number of bacteria or fungi (MPN/m3).
\(Total Colony Forming Unit\frac{CFU}{{m}^{3}}=\frac{Number ofColonies}{Air Flow rate \left(\frac{m3}{{min}}\right)*Sampling time \left(min\right)}\) (Eq. 1)
2.3 Data Analysis
The data analysis was performed using Microsoft Excel to analyse the total concentration of bacteria or fungi in response to seasonal variation at selected sampling site. We also investigated the effect of different parameters such as number of occupants, ventilation, cleanliness, biological wastewater treatment experiment, toxicity analysis on the number of bacterial or fungal count by performing corelation analysis.
3.1 Variability of Bioaerosol counts with two air sampling volumes
The air sampling volume is very important parameter to determine the colony forming unit (CFU) and get fair idea about bioaerosol concentration (CFU/m3) prevailing in indoor or outdoor environment. The standard error in MPN calculations increases with higher number of colonies counts on the petri dish. The author (Kalogerakis et al., 2005) observed that the error is amplified by a factor between 1 and 20 depending on the sample size. Theoretically, the optimum measurements should be obtained at higher sample volumes; however, the masking effect becomes significant at higher CFU units. In our study, we conducted comparative analysis of bacterial and fungal bioaerosol concentration with reference to two air sampling volume − 100 L and 200 L. The Fig. 1 indicated the mesophilic (Fig. 2a), psychrophilic (Fig. 2b) and Fungal (Fig. 2c) concentration in May month. It is clear from the graph that, among these two volumes, 100 L sampling volume is optimum for collecting existing bioaerosol inside the laboratory. At higher sampling volume, the mesophilic and psychrophilic bacteria showed reduction in CFU count. When (Kalogerakis et al., 2005) three different air sampling was performed at 200 L, 400 L and 800 L from the office building of Chania campaign, Greece, the highest number of CFU were observed at 200 L of air.
Figure 2 also indicated that the average mesophilic bacterial concentration is higher in 229a (407.04 MPN/m3) and 231a lab locations (398.22 MPN/m3). It corelate with the greater number of people working in laboratory during office hours. At 229c location, the mesophilic and psychrophilic bacteria found comparatively in less concentration, but it showed highest fungal concentration (30.15 MPN/m3). It was also observed that at this particular time the experimental culture media used in other studies were also getting highly fungal contamination. What is important to mention that the fungal morphology was found to be same of bioaerosol fungi and fungal contamination present in culture media. It indicated the laboratory air contamination with fungus, probably propagating from 229c laboratory location.
3.2 Total bioaerosol concentration
As was previously indicated, Poland does not have the required documentation to determine what levels of bioaerosol are acceptable. Legislation establishing microbiological criteria for air pollution hasn't been devised or put into place in many other nations either. This is mostly due to the wide range of air microflora, considerable changes in the intended use of the rooms and the attainable level of cleanliness in each, as well as environmental factors, or the influence of outside sources on the quality of indoor air. Another factor is related to the wide range of approaches. As a result, Poland lacks any commonly acknowledged standards for determining exposure to biological agents.
Several national and international agencies have proposed standards/guidelines for tolerable bioaerosol concentration as part of attempts to reduce the harm caused by bioaerosol exposure in indoor spaces. For instance, the World Health Organization (WHO) suggested that as a residential guideline, fungal bioaerosol concentrations of no more than 500 colony-forming units (CFU)/m3 are appropriate (Taylor et al., 2012; WHO, 1988). According to Lenart-boron et al. (Lenart-boron, 2019), the allowable level of airborne microbes in Polish zoos is 105 CFU/m3. At multi-use facilities like hospitals, healthcare centers, and daycare centers, the Ministry of Environment of the Republic of Korea mandated and advised total concentrations of bacterial (< 800 CFU/m3) and fungal (< 500 CFU/m3) bioaerosols (Kim et al., 2017). In our study, the total concentration of bacteria (800 CFU/m3) and fungi (500 CFU/m3) in bioaerosols has been set as the upper limit for indoor environments based on various guidelines. Concentrations above this level have been deemed as potent contamination that may have a negative impact on the occupants of the space.
The Table 2, Table 3 and Table 4 respectively give details of average concentration of mesophilic bacteria, psychrophilic bacteria and culturable fungi as a measure of airborne microbiota detected during the Summer (23/05/2022 to 15/06/2022), Autumn (19/09/2022 to 10/10/2022) and Early Winter (14/11/2022 to 05/12/2022) time period. By observing the number of all three biological variants of bioaerosol, it was concluded that the mesophilic and psychrophilic bacteria gave similar pattern of changes at all selected sampling sites and gave similar response to variables such as number of occupants, biological activities and seasonal changes. While the culturable fungi shows different pattern in total number changes. Overall, the concentration of mesophilic bacteria was detected in lab premises ranged from 61 to 2018 MPN/m3. The lowest mesophilic and psychrophilic bacterial concentration was observed in the site 229c during summer. In early winter, it exceeded the higher permissible limit in indoor environments. In this time, it ranged from 811 to 2018 MPN/m3. Likewise, psychrophilic bacteria also show elevated levels during the first and second week of early winter measurement period and fungi concentration was not increased.
In our study, it can be seen that the concentration levels of airborne bioaerosols are below the proposed standards in most of cases of bacterial, while fungal aerosol does not exceed this boundary. In an indoor laboratory, psychrophilic bacteria were detected in concentrations between 75 and 1130 MPN/m3, whereas fungi were found in concentrations between 10 and 434 MPN/m3. However, according to research by the Occupational Health and Safety Research Institute Robert Sauvé (IRSST), total airborne bacteria concentrations above 1000 CFU/m3 are indicative of potential microbial contamination and warrant further examination of the issue as well as the need for action (Bragoszewska et al., 2016). Although the limit has not been reached in the case of fungi, control measures are nevertheless necessary to lessen the risk of contamination for continued research. Concentrations above 500 CFU/m3 do not require corrective action, although these levels may be a sign of building-related bioaerosol sources, inadequate ventilation, or overcrowding (Pegas et al., 2010). However even with a wide range of species, the CMHC (Canada Mortgage and Housing Corporation) (Górny et al., 2004) believes that there shouldn't be more than 200 CFU/m3 of fungi. In this case, it should be stated that the air quality in all rooms was definitely good only in winter.
|
Season |
Time (Week) |
Average Concentration (MPN/m3 ± SD) at Laboratory Location |
||||
|
229a |
229b |
229c |
231a |
231 |
||
|
Summer (23/05/2022 to 15/06/2022) |
1st |
407 ± 112.2 |
278 ± 95.1 |
152 ± 17.1 |
398 ± 58.0 |
215 ± 22.6 |
|
2nd |
83 ± 33.3 |
68 ± 10.9 |
85 ± 54.4 |
184 ± 57.3 |
264 ± 12.2 |
|
|
3rd |
350 ± 19.9 |
137 ± 30.7 |
194 ± 74.4 |
1074 ± 149.1 |
491 ± 73.9 |
|
|
4th |
1178 ± 194.8 |
1286 ± 81.1 |
296 ± 55.8 |
1008 ± 193.9 |
61 ± 17.6 |
|
|
Autumn (19/09/2022 to 10/10/2022) |
1st |
307 ± 79.1 |
271 ± 114.4 |
959 ± 591.1 |
275 ± 113.6 |
284 ± 39.9 |
|
2nd |
435 ± 22.4 |
530 ± 120.9 |
360 ± 86.5 |
519 ± 151.3 |
792 ± 155.3 |
|
|
3rd |
404 ± 25.8 |
348 ± 50.9 |
260 ± 76.6 |
329 ± 100.2 |
596 ± 168.8 |
|
|
4th |
414 ± 81.8 |
335 ± 127.8 |
175 ± 28.3 |
244 ± 22.7 |
622 ± 392.3 |
|
|
Early Winter (14/11/2022 to 05/12/2022) |
1st |
1633 ± 348.9 |
1195 ± 288.7 |
811 ± 40.7 |
1314 ± 141.8 |
1174 ± 77.7 |
|
2nd |
2018 ± 169.5 |
990 ± 217.8 |
1471 ± 126.5 |
896 ± 134.1 |
904 ± 217.2 |
|
|
3rd |
731 ± 52.6 |
455 ± 130.5 |
668 ± 389.6 |
430 ± 87.3 |
706 ± 105.7 |
|
|
4th |
776 ± 48.9 |
556 ± 150.8 |
676 ± 222.5 |
490 ± 89.5 |
775 ± 110.2 |
|
|
Season |
Time (Week) |
Average Concentration (MPN/m3 ± SD) at Laboratory Location |
||||
|
229a |
229b |
229c |
231a |
231 |
||
|
Summer (23/05/2022 to 15/06/2022) |
1st |
288 ± 28.0 |
312 ± 109.8 |
105 ± 47.7 |
212 ± 54.3 |
310 ± 34.2 |
|
2nd |
115 ± 48.8 |
75 ± 21.9 |
89 ± 15.4 |
170 ± 34.1 |
241 ± 57.1 |
|
|
3rd |
282 ± 85.4 |
143 ± 58.4 |
167 ± 48.0 |
316 ± 38.1 |
241 ± 65.4 |
|
|
4th |
727 ± 84.2 |
819 ± 105.7 |
218 ± 63.2 |
785 ± 75.9 |
133 ± 59.4 |
|
|
Autumn (19/09/2022 to 10/10/2022) |
1st |
345 ± 100.0 |
311 ± 45.3 |
288 ± 60.9 |
337 ± 13.1 |
78 ± 46.3 |
|
2nd |
497 ± 52.0 |
422 ± 138.2 |
274 ± 67.8 |
607 ± 56.7 |
480 ± 33.8 |
|
|
3rd |
563 ± 75.3 |
496 ± 155.6 |
250 ± 69.6 |
506 ± 63.2 |
220 ± 39.6 |
|
|
4th |
271 ± 99.9 |
179 ± 34.4 |
176 ± 74.7 |
230 ± 95.3 |
296 ± 40.3 |
|
|
Early Winter (14/11/2022 to 05/12/2022) |
1st |
1092 ± 23.0 |
1130 ± 131.5 |
773 ± 127.2 |
891 ± 126.9 |
887 ± 150.5 |
|
2nd |
1128 ± 67.1 |
945 ± 437.8 |
672 ± 122.4 |
672 ± 125.9 |
601 ± 145.1 |
|
|
3rd |
957 ± 308.4 |
458 ± 114.6 |
626 ± 236.5 |
643 ± 142.4 |
723 ± 334.1 |
|
|
4th |
798 ± 102.8 |
556 ± 158.9 |
706 ± 159.4 |
714 ± 134.7 |
667 ± 145.7 |
|
|
Season |
Time (Week) |
Average Concentration (MPN/m3 ± SD) at Laboratory Location |
||||
|
229a |
229b |
229c |
231a |
231 |
||
|
Summer (23/05/2022 to 15/06/2022) |
1st |
23 ± 15.4 |
13 ± 5.8 |
30 ± 17.4 |
23 ± 5.8 |
30 ± 17.5 |
|
2nd |
65 ± 24.3 |
47 ± 15.8 |
79 ± 35.5 |
48 ± 35.4 |
46 ± 8.9 |
|
|
3rd |
110 ± 39.3 |
51 ± 13.8 |
70 ± 11.0 |
100 ± 24.3 |
89 ± 8.2 |
|
|
4th |
372 ± 81.1 |
394 ± 13.4 |
434 ± 90.9 |
190 ± 37.7 |
394 ± 55.0 |
|
|
Autumn (19/09/2022 to 10/10/2022) |
1st |
247 ± 22.8 |
199 ± 119.6 |
367 ± 59.1 |
314 ± 57.4 |
402 ± 29.3 |
|
2nd |
44 ± 3.0 |
61 ± 18.9 |
102 ± 10.4 |
233 ± 16.7 |
176 ± 46.3 |
|
|
3rd |
150 ± 10.8 |
91 ± 47.5 |
202 ± 36.5 |
184 ± 99.6 |
345 ± 26.8 |
|
|
4th |
194 ± 75.9 |
81 ± 37.2 |
166 ± 83.7 |
158 ± 24.5 |
406 ± 44.2 |
|
|
Early Winter (14/11/2022 to 05/12/2022) |
1st |
116 ± 42.3 |
74 ± 46.3 |
88 ± 25.9 |
137 ± 16.1 |
126 ± 21.8 |
|
2nd |
78 ± 33.0 |
37 ± 11.7 |
71 ± 20.5 |
50 ± 10.2 |
71 ± 10.2 |
|
|
3rd |
20 ± 5.5 |
30 ± 9.0 |
10 ± 4.4 |
25 ± 7.1 |
50 ± 14.4 |
|
|
4th |
45 ± 8.3 |
35 ± 12.5 |
58 ± 12.5 |
60 ± 3.7 |
82 ± 15.2 |
|
3.3 Corelation of bioaerosol with influencing factors
As mentioned previously that bioaerosol emission, transmission and survival depends on various physical and biological factors. Table 5 indicates the corelation between all three types of bioaerosol in different seasons with area of laboratory, ventilation, cleanliness and UV cleaners. According to best of our knowledge, no one has detected the corelation or influence of biological activities performed in biological laboratory (Table 6). We suspected that biological activities such as biological wastewater treatment activity, toxicity test or other experimental activities like centrifugation may influence bioaerosol concentration. We also hypothesized that when the biological material is piled up for many days for decontamination, then it may influence bioaerosol. Among all sampling area, the lowest level of bacterial aerosol was found in 229c location. It indicates that the autoclave room is equipped with proper ventilation system; hence, despite different activities performed in media and autoclave room, the bacterial contamination of air is lower. In summer, a relatively higher mesophilic bacterial concentration (average conc. 666 MPN/m3) was observed in 231a lab, indicating poor ventilation system in 231a lab premises. During this time, the zoo toxicity and phytotoxicity experiments were carried in this area. It may influence bacterial numbers as the positive corelation (0.39) found. Combination of different factors such as increased number of occupants, biological activities and poor ventilation system responsible for influence in bacterial concentration.
The higher number of mesophilic bacteria show connection with a greater number of occupants during this period as undergrad students were present in laboratory during these two weeks and engaged in various biological experiments such as dye decolorization using bacteria, actinomycetes or fungi. These experiments were responsible for such increase in concentration of microorganisms too. The concentration of mesophilic bacteria positively corelated with other experimental work (0.80) and number of occupants (0.75) in 231a lab. It was proven that indoor bacterial bioaerosol concentration can be strongly influenced by human activities, unlike fungi (Heo et al., 2016). Heo et al.(Heo & Lee, 2016) tested the effects of human presence on bioaerosol concentration under various conditions, including standing, moving, and talking. They discovered that moving conditions had the biggest effects on bacterial aerosol concentration, whereas fungal aerosol was unaffected by these tested parameters. This research supports our findings. Furthermore, Soto et al. (2009) discovered that in 10 interior settings on a university campus, the concentration of bacterial bioaerosol rose when people were present, suggesting that human activity may be a source of indoor bacterial bioaerosols (Soto et al., 2009). According to our findings, Bragoszewska et al. (2021) noted that the biomass analysis laboratory had the greatest average concentration of bacteria and fungi (673 CFU/m3 and 438 CFU/m3, respectively) (Brągoszewska & Pawlak, 2021). We can therefore state that biological sample area is more prone to bioaerosol emission. The areas of Polish wastewater treatment plants (WWTPs) where activated sludge post-processing and (in some plants) the mechanical purifying process were carried out had the highest concentrations of bacterial aerosol (1.2 * 103 − 2.8 * 103 CFU/m3 and 5.5 * 102 − 6.9 * 103 CFU/m3, respectively). This may be explained by the fact that mechanical treatment and post-processing of activated sludge are carried out indoors, in buildings, at the WWTPs where sampling was done (Wolany & Płaza, 2017). Thus, indoor biological activities are an important source for the production of bioaerosol.
It is noticed that size, concentration, and diversity of bioaerosol fluctuate with the people, kind of activities happening at particular place. Kallawicha et.al. (2019) observed in laboratory that the culturable fungi significantly increased with the number of staff and visible molds, whereas water leaks and culturable fungi significantly increased fungal spore concentrations. Culturable bacteria were positively associated with the numbers of trash bins and − 80 ℃ freezers (Kallawicha et al., 2019). It may have chances to contaminate the ongoing experiments because of high number of bioaerosols (S. Hwang et al., 2019). In one recent study of bioaerosol from an indoor environment operated wastewater management facility, they reported the dominant isolated genera of opportunistic pathogenic bacteria: Escherichia coli, Bacillus cereus, Bacillus subtilis and Pseudomonas sp. with % dominance as 38.46, 13.46, 9.61 and 25, respectively.
Table 5. Corelation of bioaerosol concentration with Area, cleanliness, Ventilation and UV cleaners in biological laboratory
|
229a |
|||||
|---|---|---|---|---|---|
|
Mesophiles |
Psychrophiles |
Culturable Fungi |
Bioremediation of wastewater |
People movement at/just before time of bioaerosol measurement |
|
|
Mesophiles |
1.00 |
||||
|
Psychrophiles |
0.88 |
1.00 |
|||
|
Culturable Fungi |
0.09 |
-0.08 |
1.00 |
||
|
Bioremediation of wastewater |
0.49 |
0.47 |
0.03 |
1.00 |
|
|
People movement at/just before time of bioaerosol measurement |
0.71 |
0.47 |
0.33 |
0.60 |
1.00 |
|
229b |
|||||
|---|---|---|---|---|---|
|
Mesophiles |
Psychrophiles |
Culturable Fungi |
Bioremediation of wastewater |
People movement at/before time of bioaerosol measurement |
|
|
Mesophiles |
1.00 |
||||
|
Psychrophiles |
0.93 |
1.00 |
|||
|
Culturable Fungi |
0.48 |
0.25 |
1.00 |
||
|
Bioremediation of wastewater |
0.40 |
0.48 |
0.07 |
1.00 |
|
|
People movement at/before time of bioaerosol measurement |
0.69 |
0.67 |
0.09 |
0.17 |
1.00 |
|
229c |
|||||
|---|---|---|---|---|---|
|
Mesophiles |
Psychrophiles |
Culturable Fungi |
content for decontamination complied for many days |
People movement at/before time of bioaerosol measurement |
|
|
Mesophiles |
1.00 |
||||
|
Psychrophiles |
0.76 |
1.00 |
|||
|
Culturable Fungi |
-0.01 |
-0.31 |
1.00 |
||
|
content for decontamination complied for many days |
0.25 |
0.20 |
-0.28 |
1.00 |
|
|
People movement at/before time of bioaerosol measurement |
0.71 |
0.59 |
0.42 |
0.12 |
1.00 |
|
231a |
||||||
|---|---|---|---|---|---|---|
|
Mesophiles |
Psychrophiles |
Culturable Fungi |
Toxicity Test |
Other experimental work (centrifugation, spectrophotometry, washing etc.) |
People movement at/before time of bioaerosol measurement |
|
|
Mesophiles |
1.00 |
|||||
|
Psychrophiles |
0.63 |
1.00 |
||||
|
Culturable Fungi |
-0.05 |
0.05 |
1.00 |
|||
|
Toxicity Test |
0.39 |
0.18 |
-0.14 |
1.00 |
||
|
Other experimental work (centrifugation, spectrophotometry, washing etc.) |
0.80 |
0.24 |
-0.26 |
0.29 |
1.00 |
|
|
People movement at/before time of bioaerosol measurement |
0.75 |
0.31 |
-0.05 |
0.19 |
0.85 |
1.00 |
3.4 Effect of Seasonal Variation on concentration of Bioaerosol
The airborne microflora has direct relation with temporal parameters. It changes with reference to change in temperature, humidity etc. (Heo & Lee, 2016). Along with outdoor air, the indoor air microbial diversity is also influenced by seasonal changes. We inspected the effect of seasonal variation on the total concentration of indoor culturable bioaerosol. These data represent analysis of bioaerosol during three seasons in Poland: Summer, Autumn and Early Winter. The temperature and relative humidity fluctuations were recorded each time during sampling (Fig. 4). It indicates the highest temperature recorded in Summer- approximately 25℃, whereas the highest relative humidity was observed in Autumn- approximately 55%. During Autumn time, the combination of temperature and humidity provide suitable weather for growth and propagation of fungi.
Heo et.al. (2016) confirmed the bacterial aerosol concentration in the subway systems varied throughout the seasonal transitions from spring to summer and from fall to winter (Heo & Lee, 2016). As well we found the highest concentration of bacterial aerosol in early wintertime (Fig. 3a, Fig. 3b). As mentioned previously that during this time, a more significant number of occupants activity was found in lab locations 229a and 231a. So, it confirms the influence of occupants’ activity but as well season influence on average concentration of mesophilic and psychrophilic bacteria.
Environmental conditions influence fungal air microbiota. The highest concentration of fungal aerosol was observed in Autumn time (Fig. 3c) where temperatures is relatively lower and greater humidity exist (Fig. 4). From all selected locations, the greater number of fungal aerosol was found in 231a lab and 231 (235 MPN/m3). In summer, all locations of 229 laboratory were contaminated with fungal aerosol whereas early wintertime showed reduction in fungal aerosol at all locations. Although these numbers are below permissible limits of indoor fungal bioaerosol concentrations, they need to be monitored and eliminated to protect employees and students from fungal exposure.
3.5 Species richness of each microbial group
Bioaerosol study has directly relates to workers’ health risks as some of the bacteria and fungi found in indoor environments are pathogenic. They can live in human skin and hair for longer and spread to nearby places. Hence, it is emphasized to study the microbial diversity of indoor air, along with the study of the concentration of bacterial and fungal bioaerosol. The types of fungi characteristic to closed environments are Alternaria sp., Aspergillus sp., Cladosporium sp., Fusarium spp. and Penicillum sp. etc. Humid air is highly conducive to the development of fungi. The presence of fungus like Aspergillus sp. in microbiology can be a potent contamination for ongoing experiments in laboratory. It is also important to study mesophilic and psychrophilic bacterial concentration in air (Haas et al., 2020). Among bacteria in the internal air, dominant species include Micrococcus sp. and Staphylococcus epidermidis (staphylococci colonizing human skin). Another genus is also present such as Bacillus, Enterobacter, Flavobacterium, Pseudomonas and Streptomyces. We observed colony characteristics of bacteria and fungi as shown in Fig. 5. Staphylococcus aureus is known to produce pigmented or non-pigmented colonies on TSA plates. The staphyloxanthin is what gives yellow pigment. This pigment serves as a virulence factor by assisting bacteria in avoiding reactive oxygen species, which the host immune system uses to destroy pathogens (Han, Yang, et al., 2020). Staphylococcus bacteria make up the natural microflora of human skin and mucosa. Both those with healthy immune systems and individuals with compromised immune systems are at risk from them. Antibiotic-resistant strains could be very harmful.
We focused on studying the distribution of bioaerosol on Tryptic soy agar plates. Antibiotics were not included. Hence, it allowed the growth of a range of microbial species diversity. The observation wad divided into three categories based on pigmented colonies, non-pigmented colonies and filamentous fungal colonies and/or mold. On TSA plate, incubated at higher temperatures (37 ℃), the fungal colonies constitute very less portion. These plates are dominated by pigmented bacterial colonies followed by non-pigmented bacterial colonies. In early winter, the yellow or pink pigmented colonies dominated by approx. 70%.
On TSA plate, incubated at lower temperatures (20 ℃), the significant proportion of fungal or mold were observed, especially in autumn time (Fig. 7). The generation distribution order was observed as fungal colonies > pigmented bacterial colonies > non-pigmented bacterial colonies. When morphological types were observed for bacterial and fungal colonies based on colony morphology, it showed great diversification of bioaerosol composition in specific air volume (Fig. 6 and Fig. 7). The abundance and diversity of fungal bioaerosol increased during Autumn time. Ontiveros et. al. (2021), developed a predictive method that dynamically addressed the temporal evolution of biodiversity in response to environmental covariates, linked to future climatic scenarios. They discovered microbiota general decline in airborne microbiota diversity under future climatic conditions (Ontiveros et al., 2021). They emphasized unravelling air microbiota composition. As indoor air composition less frequently changes compared to outdoor air, the diversity study of indoor air is of utmost importance. If one kind of microbial species is found in indoor environment, it is detrimental to the health of occupants because of the majority of any one microbial species. For example, If Aspergillus spp. dominates in indoor air, the people can breathe its spores, which are responsible for Aspergillosis disease.
Our studies indicated that bioaerosol formation inside biological laboratories is influenced by various parameters such as occupants’ number, their activity, ventilation system and seasonal changes. And it is get influenced by different biological activities also. Although the total bioaerosol concentrations were studied during this research, indicating that culturable bacteria were slightly more than permissible limits and fungi concentrations were falling within limits. Still the control measures needed to control bioaerosol concentrations inside university laboratories as the culturable microbiota does not exactly represent the number of bioaerosols. The actual number of microorganisms exceeds the number of culturable bioaerosols. It indicates the scope of future research on the next generation sequencing approach with suitable bioinformatics to study bioaerosol diversity.
Acknowledgement: The authors thank the Silesian University of Technology, Poland and Karunya Institute of Technology and Sciences Coimbatore, TN, India for support and help for submitted research.
Funding:
The authors would like to acknowledge the Faculty of Energy and Environmental Engineering, Silesian University of Technology Poland to provide a grant (BKM 676/RIE2/2022, 08/020/BKM22/0027) to support research activities of young scientists.
- Competing Interest:
The authors declare that they have no known competing financial interests or personal relationships that influence the work reported in this paper.
- Authors’ contribution statement:
Ruchi Upadhyay: Conceptualization; Investigation; Data curation; Formal analysis; Funding acquisition; Methodology; Visualization; Validation; Writing original draft.
Wioletta Przystaś: Conceptualization; Supervision; Writing - review & editing.
Sneha Gautam: Writing - review & editing.
- Biologiczne czynniki szkodliwe: normy, zalecenia i propozycje warto ś ci dopuszczalnych. (2004). 3(3), 17–40.
- Br, E., Mainka, A., & Pastuszka, J. S. (2016). Bacterial and Fungal Aerosols in Rural Nursery Schools in Southern Poland. https://doi.org/10.3390/atmos7110142
- Brągoszewska, E. (2019). Exposure to bacterial and fungal aerosols: Microorganism indices in a waste-sorting plant in Poland. International Journal of Environmental Research and Public Health, 16(18). https://doi.org/10.3390/ijerph16183308
- Brągoszewska, E., & Pawlak, M. (2021). Health risks associated with occupational exposure to biological air pollutants occurring during the processing of biomass for energy purposes: A case study. Energies, 14(8). https://doi.org/10.3390/en14082086
- Dubuis, M., Duchaine, C., & Bennett, A. (2021). Aerosol Production During Blood and Urine Pre-analytical Processing and Handling in a Hospital Biochemistry Clinical Laboratory During the COVID-19 Pandemic. 9(April), 1–5. https://doi.org/10.3389/fpubh.2021.643724
- Haas, D., Kriso, A., Fritz, T., Galler, H., Habib, J., Ilieva, M., Kropsch, M., Ofner-kopeinig, P., Stonitsch, M., Strasser, A., Zentner, E., & Reinthaler, F. F. (n.d.). Background Concentrations of Cultivable, Mesophilic Bacteria and Dust Particles in the Air in Urban, Rural and Mountain Regions.
- Han, Y., Li, L., Wang, Y., Ma, J., Li, P., Han, C., & Liu, J. (2020). Composition, dispersion, and health risks of bioaerosols in wastewater treatment plants: A review. Frontiers of Environmental Science and Engineering, 15(3), 1–16. https://doi.org/10.1007/s11783-020-1330-1
- Han, Y., Yang, T., Xu, G., Li, L., & Liu, J. (2020). Characteristics and interactions of bioaerosol microorganisms from wastewater treatment plants. Journal of Hazardous Materials, 391, 122256. https://doi.org/10.1016/j.jhazmat.2020.122256
- Heo, K. J., & Lee, B. U. (2016). Seasonal variation in the concentrations of culturable bacterial and fungal aerosols in underground subway systems. Journal of Aerosol Science, 92, 122–129. https://doi.org/10.1016/j.jaerosci.2015.11.004
- Heo, K. J., Lim, C. E., Kim, H. B., & Lee, B. U. (2016). Author ’ s Accepted Manuscript. Journal of Aerosol Science. https://doi.org/10.1016/j.jaerosci.2016.11.008
- Hwang, S. H., Park, D. U., & Yoon, C. S. (2017). Human and Ecological Risk Assessment: An International Levels of airborne biological agents and related factors in indoor environments of fish toxicity laboratory. 7039. https://doi.org/10.1080/10807039.2017.1321955
- Hwang, S., Ko, Y., Park, D., & Yoon, C. (2017). Seasonality in airborne bacterial, fungal, and (1 → 3) - β -D-glucan concentrations in two indoor laboratory animal rooms. 1–8. https://doi.org/10.1136/jclinpath-2017-204377
- Hwang, S., Park, D. U., & Yoon, C. (2019). Seasonal variation in (1→3)-β-D-glucan levels with health risk assessment and related factors in indoor environments of microbiology laboratories. Human and Ecological Risk Assessment, 25(5), 1096–1106. https://doi.org/10.1080/10807039.2018.1460578
- Jureczko, M., Przystaś, W., Krawczyk, T., Gonciarz, W., & Rudnicka, K. (2021). White-rot fungi-mediated biodegradation of cytostatic drugs - bleomycin and vincristine. Journal of Hazardous Materials, 407(July 2020). https://doi.org/10.1016/j.jhazmat.2020.124632
- Kallawicha, K., Chao, H. J., & Kotchasatan, N. (2019). Bioaerosol levels and the indoor air quality of laboratories in Bangkok metropolis. Aerobiologia, 35(1), 1–14. https://doi.org/10.1007/s10453-018-9535-5
- Kalogerakis, N., Paschali, D., Lekaditis, V., Pantidou, A., Eleftheriadis, K., & Lazaridis, M. (2005). Indoor air quality - Bioaerosol measurements in domestic and office premises. Journal of Aerosol Science, 36(5–6), 751–761. https://doi.org/10.1016/j.jaerosci.2005.02.004
- Kim, K., Kabir, E., & Jahan, S. A. (2017). Airborne bioaerosols and their impact on human health. Journal of Environmental Sciences, 1–13. https://doi.org/10.1016/j.jes.2017.08.027
- Lenart-boron, J. G. A. (2019). Bacterial bioaerosol concentration and size distribution in the selected animal premises in a zoological garden. 9, 253–268. https://doi.org/10.1007/s10453-018-09557-9
- Michałkiewicz, M., Kruszelnicka, I., & Widomska, M. (2018). The variability of the concentration of bioaerosols above the chambers of biological wastewater treatment. Ecological Chemistry and Engineering S, 25(2), 267–278. https://doi.org/10.1515/eces-2018-0018
- Ontiveros, V. J., Cáliz, J., Margarit, X. T., Alonso, D., & Casamayor, E. O. (2021). General decline in the diversity of the airborne microbiota under future climatic scenarios. Scientific Reports, 0123456789, 1–7. https://doi.org/10.1038/s41598-021-99223-x
- Pegas, P. N., Evtyugina, M. G., Alves, C. A., Nunes, T., Cerqueira, M., & Franchi, M. (2010). Artigo. 33(5), 1145–1149.
- Singh, N. K., Sanghvi, G., Yadav, M., Padhiyar, H., Gupta, A., Christian, J., & Thanki, A. (2022). Assessment and characterization of bioaerosols from an indoor environment-operated wastewater management facility: unraveling pathogenicity in research laboratories. Aerobiologia, 0123456789. https://doi.org/10.1007/s10453-022-09763-6
- Soto, T., Murcia, R. M. G., Franco, A., Vicente-soler, J., Cansado, J., & Gacto, M. (2009). Indoor airborne microbial load in a Spanish university (University of Murcia, Spain). 109–115.
- Taylor, P., Rao, C. Y., Burge, H. A., Chang, J. C. S., Rao, C. Y., & Burge, H. A. (2012). Review of Quantitative Standards and Guidelines for Fungi in Indoor Air Review of Quantitative Standards and Guidelines for Fungi in Indoor Air. September 2014, 37–41. https://doi.org/10.1080/10473289.1996.10467526
- Upadhyay, R., & Przysta, W. (2022). DECOLORIZATION OF TWO DYES USING WHITE ROT FUNGUS. OSTREATUS (BWPH) STRAIN AND EVALUATION OF ZOOTOXICITY. 87–94. https://doi.org/10.2478/ACEE-2022-0033
- WHO_SDE_OEH_99.14.pdf. (n.d.).
- Wolany, M. K. J., & Płaza, J. S. P. G. (2017). Characteristics of airborne bacteria and fungi in some Polish wastewater treatment plants. International Journal of Environmental Science and Technology, 14(10), 2181–2192. https://doi.org/10.1007/s13762-017-1314-2
- Wolny-ko, K., & Malinowski, M. (2017). I ndoor and Built Microbiological air contamination in university premises and the evaluation of drug resistance of staphylococci occurring in the form of a bioaerosol. 0(0), 1–12. https://doi.org/10.1177/1420326X17748463
No competing interests reported.