Utilising high-throughput sequencing, this study assessed the bacterial communities of bioaerosols emitted from two municipal WWTPs that use different aeration modes. The findings shed light on a broad profile of bacteria, including potential opportunistic pathogenic bacteria, present in the air at WWTPs. The results of this study demonstrated that bacterial composition of bioaerosols generated by MAER was diverse when compared to bioaerosols generated by DAER. These findings are further supported in a recent study where significantly higher bacterial diversity was observed in bioaerosols produced by MAER than the background air²⁷. The study further indicated that rotor speed influences species composition, size distribution, and concentration of bioaerosols, and that the levels of bacteria in the air increased with accelerated rotational speed²⁷. The secondary treatment step is a key component of the wastewater treatment process, aimed at biologically removing organic components in wastewater²⁸. Mechanical (e.g. rotors, fountains, agitators and others) and diffused (e.g. fine and coarse bubble diffusers) aerator systems are commonly utilised to aerate wastewater in aeration tanks, resulting in bioaerosol emission due to intense mixing and turbulence. The type of aeration system used determines the size of bubbles formed, which in turn influences the size of the bioaerosol and its microbial load^16,22. Additionally, distinct genera were identified in the background controls compared to aeration tanks samples. Differences in bacterial composition across different treatment stages in WWTPs are primarily influenced by factors including mechanical processes (e.g., aeration and agitation), meteorological conditions (e.g., temperature, humidity, solar radiation, and wind), and sources of bacteria (e.g., wastewater, sludge, and background atmospheric sources)^13,15. Therefore, it is plausible that bacteria from other sources unrelated to wastewater may have been dominant in the background control samples, contributing to the observed differences in bacterial species.

Although the bioaerosol data in this study are presented as relative abundances rather than quantitative measurements, the inclusion of environmental metadata provides a valuable context for interpreting the results. Environmental factors such as relative humidity, temperature, wind and UV index are known to influence bioaerosol dynamics and could partially explain the observed shifts in relative abundance^13,15. For example, a study investigating bioaerosols from six municipal WWTPs in China demonstrated that meteorological factors, particularly temperature, solar radiation, and relative humidity influence the survival and dispersion of airborne microorganisms²⁹. Temperature and relative humidity directly affect bioaerosol dispersal and viability of airborne microorganisms, thus affecting the concentration of bioaerosols. Wind dilute and reduce bioaerosol concentrations, whereas poor ventilation may lead to significant accumulation of microbial aerosols in indoor treatment units^13,15. Since the environmental metadata is averaged over a larger area, it may not perfectly represent microclimatic conditions at the study site. However, the data provides a reasonable approximation of the prevailing environmental conditions at the studied areas during the sampling period.

In this study, majority of potentially opportunistic pathogenic bacterial genera and species were identified in MAER bioaerosols. These findings are comparable with previous studies that explored bacterial composition in bioaerosols from WWTPs using different aeration modes^16,22,27. Intestinal bacteria in bioaerosols from six municipal WWTPs in China were substantially higher in mechanical aerators than biochemical reaction tanks utilising diffused aerators¹¹. Similarly, Han and co-workers (2020) indicated that mechanical aerators (horizontal rotors) contributed the most total microorganisms and specific pathogens in bioaerosols when compared to fine bubble diffusers in the same WWTP²². In a recent study, Lu et al.⁴ collected air samples from the grit chamber house and near the aeration tank at a municipal WWTP in Denmark, identifying 91 and 94 bacterial species, respectively, with five Risk Group 2 species (Enterobacter cloacae, Klebsiella oxytoca, K. pneumoniae, Escherichia coli, and Morganella morganii) present at both sites.

Potentially pathogenic genera in this study were dominated by Bacillus spp., Enterococcus spp., Clostridium spp., Streptococcus spp., Acinetobacter spp., Enterobacter spp., Pseudomonas spp., Bacteroides fragilis, Acinetobacter baumannii, and Escherichia/Shigella. These bacterial species can cause skin, community acquired pneumonia, gastrointestinal, and urinary tract infections (UTI) in humans. Previous studies have found similar pathogenic bacteria in bioaerosols from WWTPs^{4,11,12,13,16,17}. Bacillus species are common in soil and wastewater, and the species of medical importance are Bacillus anthracis and Bacillus cereus, which are responsible for anthrax and food poisoning, respectively³⁰. Enterococcus species can cause UTI, meningitis, infective endocarditis (IE), and wound infections³¹. Clostridial infections, on the other hand, are typically associated with food poisoning, gas gangrene, botulism, and soft tissue infections³². Acinetobacter species can cause infections of the respiratory tract, blood and urinary tract, particularly in immuno-compromised individuals. Of note, A. baumannii is one of the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp.) pathogens primarily associated with hospital-acquired infections as well as antimicrobial resistance³³. Streptococcus species can cause invasive diseases such as sepsis, meningitis, IE, and pneumonia³⁴, whereas the Enterobacter genus is a well-known nosocomial pathogen that causes UTI, osteomyelitis, IE, respiratory, and soft tissue infections³⁵. Pseudomonas aeruginosa causes the majority of Pseudomonas infections, and can infect virtually any tissue, including the blood, central nervous system, and lungs, among others³⁶. Bacteroides fragilis can cause peritonitis, lung and brain abscesses, bacteremia, soft tissue infections, and toxin-associated diarrhoea³⁷. Common infections of Escherichia species include UTI, pneumonia, bacteremia, and acute enteritis³⁸, whereas Shigella is a well-known cause of acute gastrointestinal infections³⁹.

Twenty-seven human bacterial pathogens identified in this study are classified as Risk Group 2, whereas Escherichia/Shigella belong to Risk group 2 or 3²⁶. Overall, this study suggests that WWTP workers are potentially exposed to airborne bacterial pathogens that can cause a range of diseases such as respiratory and gastrointestinal infections, UTI, and skin and soft tissue infections. Five species (E. faecium, A. baumannii, K. pneumoniae, P. aeruginosa, and Enterobacter spp.) that are highly virulent and associated with multidrug resistance were also identified. Furthermore, a majority of the classified bacterial pathogens were gram-negative bacteria, suggesting that workers at WWTPs may be exposed to higher levels of inhalable endotoxins present in their ambient air, potentially leading to chronic diseases such as bronchitis, asthma, wheeze, and organic toxic dust syndrome after long-term exposure⁴⁰. This highlights the importance of effective bioaerosol control strategies to safeguard the health of WWTP workers. This could include engineering controls such as covering aeration tanks, particularly at WWTPs using mechanical aerators, to minimise bioaerosol generation and reduce the concentration of bioaerosols in the air. In addition, implementing worker exposure controls such as strict adherence to PPE and administrative measures, such as work schedule adjustments and regular training, can further mitigate the risk of bioaerosol exposure among WWTP workers.