Obtained concentration levels of bacterial and fungal aerosols are presented in Table 4.
It can be seen that the levels of airborne fungi were very similar to those registered at the background location (102–103 CFU/m3), unaffected by the activities taking place in the wastewater treatment plants. Furthermore, similar, quite high concentrations were obtained for backgrounds (outside these plants).
In the most of the studied plants, also the concentration of airborne bacteria was comparable with the background bioaerosol. The same result was previously reported by Sánchez-Monedero et al. (2008). Only in the all stages of the WWTP "Przyjaźń", the concentration levels of bacterial aerosol were significantly elevated compared to the background, but it should be remarked that the background level was very low there.
As presented, the highest concentration of bacterial aerosol was found in these parts of wastewater treatment plant, where activated sludge post-processing and (in some plants) process of mechanical purifying were conducted (1.2 × 103–2.8 × 103 CFU/m3 and 5.5 × 102–6.9 × 103 CFU/m3, respectively). This may be referred to fact that the process of mechanical treating and post-processing of activated sludge on WWTPs, where sampling was carried out, is conducted indoor, in the buildings, e.g., of screened solids or hydraulic presses of the sludge. Concentrations of bacterial aerosol emitted in stage of biological reactors and clarifiers were in the level of 102 CFU/m3. Similar results were reported by others. Results obtained in case of samples from different stages of municipal WWTP in Spain shown that the highest amount of heterotrophic bacteria (5.6 × 103 CFU/m3) were noted in pretreatment section, while in other parts were significantly lower (2.2 × 103 CFU/m3 in primary clarifiers and 5 × 102 CFU/m3 in aeration basins) (Pascual et al. 2003). For example, Karra and Katsivela (2007) found that in Greece the highest concentrations of airborne microorganisms were observed at the aerated grit removal of wastewater at the pretreatment stage (mesophilic heterotrophic bacteria: 933 ± 636 CFU/m3; fungi: 380 ± 200 CFU/m3) than in indoor control (bacteria: 515 ± 295 CFU/m3; fungi: 160 ± 50 CFU/m3). Wlazło et al. (2002) studied distribution of the exposure to airborne bacteria inside the small wastewater treatment plant in Myszków, Poland, obtaining the concentration level of total bacteria between 102 and 103 CFU/m3 and indicated that the highest level was near the aeration basin. Bauer et al. (2002) also found in wastewater treatment plants in Austria (where the averaged concentrations of mesophilic bacteria and fungi were 1.7 × 104 and 1.7 × 103 CFU/m3, respectively) the highest exposure to bacterial and fungal aerosols during aeration. Korzeniewska et al. (2009) studied emission of airborne microorganisms from WWTP with bioreactor “BIO-PAK” found that the highest concentrations of bacteria (101–103 CFU/m3) and fungi (104 CFU/m3) were determined in air sampled inside the bioreactor, in its vicinity, and near the great chamber. It is interesting to note that in the studies concerning exposure to bioaerosol from sewage systems in Austria, the highest concentrations of mesophilic bacteria were found in the encased grit chamber. During high-pressure cleaning, total bacterial concentrations reached up to 4.0 × 104 CFU/m3, including coliforms (up to 3.0 × 103 CFU/m3) (Haas et al. 2009).
Generally, the distribution of the airborne bacterial levels in the area of the studied waste water treatment plants agrees well with the existing knowledge (Sánchez-Monedero et al. 2008; Fracchia et al. 2006). In particular, we confirmed that splashing and bubble bursting that occur as a result of forced aeration in activated sludge processes release large amount of bacteria into the air.
Analysis of the number and aerodynamic diameter of viable microorganisms collected on different stages of the impactor in the all studied wastewater treatment plants is presented in Figs. 2 and 3. It can be seen that the size distributions of airborne bacteria in the section of the clarifiers and the sludge post-processing have the peak in the size range between 2.1 and 3.3 µm while in the section of mechanical treatment, as well as in the aeration tanks the peak appears in the size range 3.3–4.7 µm, i.e., is shifted into larger particles. These results agree well with the other reports, especially with the data published by Laitinen et al. (1994) who found that most of the bacteria-carrying particles in the air of a WWTP had an aerodynamic diameter below 4.7 µm.
It is interesting to note that the patterns of the size distributions of airborne fungi are generally very similar to bacterial size distributions but have the peaks, typically, in the size range between 2.1–3.3 and 3.3–4.7 µm. This is typical size distribution pattern for airborne fungi, and similar results have been obtained in highly moldy homes in Poland (Pastuszka et al. 2000, 2005). As it can be seen the contribution of fine fungal particles (less than 2.1 µm) is significant in the section of the mechanical treatment and aeration tanks.
On the other hand, it should be noted that bioaerosol particles less than 4.7 µm should be classified as the relatively small, respirable particles. The dominating mode of small airborne bacteria and fungi in the sampling sites which may indicate that the studied bioaerosol is relatively fresh, and mostly local origin.
Percentage composition of bacterial aerosols sampled in the studied WWTPs is shown in Fig. 4. It can be seen that among the isolated bacterial species the following two groups occurred more frequently than others: Gram-positive cocci and nonsporing Gram-positive rods. Main sources of emission of Gram-positive cocci are human organisms, but also the environment (soil, water), especially in case of Micrococcus/Kocuria species. As nonsporing Gram-positive rods can be frequently found in soil, plants, water, sewage, etc., they are also typical bacteria in the wastewater treatment plant environment. Share of the other bacteria groups, involving endospore-forming Gram-positive bacilli, mesophilic actinomycetes, and Gram-negative rods, was 1/4 of the total bacterial aerosols in the studied plants.
Detailed analysis of the isolated bacteria indicates occurrence of 21 species, belonging to 11 from the genera. The most frequently occurring were bacteria types Staphylococcus (6 species) and Bacillus (3 species). Among them, the following bacterial species could be identified:
Gram-positive cocci: Staphylococcus gallinarum, Staphylococcus lentus, Staphylococcus xylosus, Kocuria rosea, Staphylococcus sciuri, Staphylococcus auricularis, Micrococcus luteus, Micrococcus spp., Kocuria varians, Staphylococcus cohnii; Nonsporing Gram-positive rods: Brevibacterium spp., Microbacterium spp., Rothia mucilaginosa, Corynebacterium spp.; Endospore-forming Gram-positive bacilli: Bacillus firmus, Bacillus mycoides, Bacillus cereus; Mesophilic actinomycetes: Streptomyces spp., Nocardia spp.; Gram-negative rods: Pseudomonas spp., Pseudomonas stutzeri.
It should be mentioned that in the wastewater treatment plants “Myszków,” in 2002 the Gram-negative bacteria contributed about 35% to the total bacterial aerosol, while in this study, their contribution did not exceed 8.5% what can indicate that the hygienic and technological advancement has been improved in Polish WWTPs during the last decade (Wlazło et al. 2002). The reduction in Gram-negative bacteria emission should still remain as one of the purposes to improve the environmental quality in WWTPs. Other studies, reviewing the main health effects among the workers revealed that even though the case of the symptoms is unknown, the results suggested that endotoxin in Gram-negative bacteria may be one of the possible causes (Thorn and Kerekes 2001).
Obtained results concerning identification of fungi species from the wastewater treatment plant’s bioaerosol, collected in aeration tanks section, are presented in Fig. 5. Results obtained for all treatment plants show that the most frequently occurring airborne fungi were Cladosporioides species (C. herbarum and C. cladosporides). C. herbarum dominated in sampled fractions >3.3 µm, while C. cladosporides appears more often in finer fractions. In the sample “Z” also significant contribution of Candida sp. was found. In the all studied wastewater treatment plants, Rhodotorula sp. was also found in the fungal aerosol. Other identified species were: Mycelia sterilia complex, and occasionally Penicillium sp. The high frequency of the occurrence of the Fusarium graminearum should be also noted.
Results of antibiotic resistance testing (Table 5) can be presented as the inhibition rate of growth diameter around antimicrobial susceptibility testing disks, mean values in case of each antibiotic and tested strain (in millimeters).
It can be seen that among the isolated species, the highest antibiotic resistance revealed Bacillus species (especially B. mycoides), what is evidenced by obtaining low growth inhibition area. On the other hand, the most antibiotic-sensitive group of bacteria (from the isolated cultures) is Kocuria sp., except the influence of quinolones, sulfonamides and nitrofurantoin. Among the tested groups of the antibiotics, the least antimicrobial activity toward airborne bacteria isolated from the WWTPs is quinolones and sulfonamides. Only few species were sensitive to them. The most effective antibiotics were penicillins, cephalosporins and aminoglycosides, which are commonly used in the antibiotic treatment.
Currently there are almost no published studies concerning the antibiotic resistance of the bioaerosols emitted in wastewater treatment plants. Research concerning the antimicrobial resistance of airborne bacteria collected in Gwalior trade fair (urban area, central India) indicated that during the fair the antibacterial resistance of the suspected strains (S. aureus, Staphylococci, Enterococcus sp., Bacillus sp., Escherichia coli and Pseunomonas sp.) increased (Yadav et al. 2013). Other studies conducted in China concerning identification of antibiotics and antimicrobial strains in soil from wastewater irrigation areas. Authors showed that even in locations, where the antibiotics were not detected, resistant strains were still observed (Chen and Zhang 2013).
The wide application of antibiotics in human and veterinary medicine has led to large-scale dissemination of bacteria resistant to antibiotics in different elements of the environment. The main sources of resistant bacteria are manure and liquid manure of animals as well as human excretions. They serve as a reservoir for bacteria with multiple resistances. Endogenous bacterial biota plays an important role as acceptor and donor of transmissible drug resistance genes. However, further investigations are required concerning effect of antibiotic resistance strains released from WWTPs on the surface waters, soil and air. Future evaluation and control are needed to evaluate and reduce public health risk.
Previous studies on antibiotic-resistant bacterial population in wastewater treatment plants have been done only in aquatic environments. Few of them have focused on the antibiotic-resistant bacteria, among them on E. coli and Acinetabacter spp. in the effluents of WWTPs and their receiving water body (Chen and Zhang, 2013; Schwartz et al. 2003; Reinthaler et al. 2003; Zhang et al. 2009; Gao et al. 2012). Reinthaler et al. (2003) evaluated resistance patterns of E. coli in wastewater treatment plants (WWTPs). Investigations have been done in sewage, sludge and receiving waters from the WWTPs. The highest resistance rates were found in the following: penicillin (ampicillin and piperacillin), cephalosporin group (cefalotin and cefuroxime), quinolone (nalidixic acid) and trimethoprime/sulfamethoxazole, and for tetracycline. Of all the investigated antimicrobial substances, the highest rate of resistance was noted for tetracycline (up to 57%). Korzeniewska et al. (2013) investigated the contamination degree of hospital effluents and municipal sewage (inflow, sewage in aeration tank, outflow) with antibiotic-resistant and beta-lactamases producing E. coli strains. E. coli strains emission to the air near selected WWTP facilities and to the river, where the treated effluent is discharged, was also determined. The results obtained by the authors indicated that antibiotic-resistant E. coli strains were emitted from sewage to the atmospheric air near WWTPs and their surroundings or directly into the water sources. Płaza et al. (2013) evaluated the antibiotic resistance of strains isolated from WWTP effluent. 90% of isolated E. coli was categorized as resistant. The highest resistance frequencies were found for the following: cefalotin and erythromycin (90%), nitrofurantoin (94%), rifampicin (97%) and novobiocin (100%).
In the present study, the collection of bacterial strains was also analyzed for multiple antibiotic resistance (MAR). The data indicated that 88% of bacteria showed resistance to 8–15 antibiotics and 3% of isolates were resistance to 19 tested antibiotics. No strain was detected to be resistance from 0 to 5 antibiotics. The decrease in susceptibility of bacterial isolates to antibiotics was probably caused by the presence of these compounds in the wastewaters and the long exposition of E. coli strains to them. Zhang et al. (2009) detected that resistance among Acinetobacter isolates to 3 antibiotics (amoxicillin, chloramphenicol and rifampin) and multi-drug resistance (more than 3 antibiotics) significantly increased from the raw influent samples to the final effluent samples.