key: cord-285725-gge8ri93
authors: Burdsall, Adam C.; Xing, Yun; Cooper, Casey W.; Harper, Willie F.
title: Bioaerosol emissions from activated sludge basins: Characterization, release, and attenuation
date: 2020-08-20
journal: Sci Total Environ
DOI: 10.1016/j.scitotenv.2020.141852
sha: 
doc_id: 285725
cord_uid: gge8ri93

Abstract This article presents a critical review of the peer-reviewed literature related to bioaerosol generation from activated sludge basins. Characterization techniques include a variety of culture- and nonculture-based techniques, each with unique features. Bioaerosols contain a variety of clinical pathogens including Staphylococcus saprophyticus, Clostridium perfringens, and Salmonella enteritidis; exposure to these microorganisms increases human health risks. Release mechanisms involve splashing and bubble burst dynamics. Larger bubbles emit more aerosol particles than smaller ones. Attenuation strategies include covering sources with lids, adjusting the method and intensity of aeration, and using free-floating carrier media. Future studies should combine culture and non-culture based methods, and expand chemical databases and spectral libraries in order to realize the full power of real-time online monitoring.

samples of bacteria and fungi lasting up to several hours (Lin et al., 1999) . The bioaerosol samples are eventually transferred onto plates or dissolved into a liquid solution for further microscopic examination, culturing experiments or non-cultured analysis such as PCR analysis. Efficiency of collection is dependent on the type of sampling method, materials of the sampler, operation parameters of the sampling devices and the microbial species (Kim et al., 2018) .

2.2.1. Culture-based techniques. Culture-based techniques are the most well-established and readily available for bioaerosol assessment. It is also a very sensitive technique and many different species can be identified without the need for specific biomarkers or primers. Culturable bioaerosols are normally sampled by using impactors (microorganisms are collected directly on a culture medium), liquid impinger (microorganisms are collected in liquid collection fluid) or air filtration methods (microorganisms are collected on a filter). The collected bioaerosols are then transferred to the lab and grown on petri dishes with a chosen media (e.g. LB, nutrient agar for bacteria; Sabouraud dextrose agar for fungi) for a few days (Yazdanbakhsh et al., 2020) . Colonies can be counted manually or with the aid of image analysis techniques. Additionally, selective culture mediums are available to allow for the culture, identification, and quantification of a species of interest while inhibiting the growth of other microorganisms (Aithinne et al., 2018; Hustá et al., 2020) . Culture-based techniques only measure the cultivable microorganisms and tend to underestimate the bioaerosol concentration due to several reasons that may cause viability loss. These factors include bioaerosol drying out in filtration-based sampling, atmospheric temperature, relative humidity, exposure to UV radiation (Douglas et al., 2017) . Cellular aggregation can also cause large variations in culture-based data.

Currently the most popular non-culture-based method is the polymerase chain reaction (PCR) method which detect and identify microorganisms and viruses by DNA or RNA sequence comparison. A genetic sequence representing a specific microorganism is first targeted J o u r n a l P r e -p r o o f Journal Pre-proof by a carefully designed DNA primer, then amplified, quantified, and sequenced. The resulting sequences can then be analyzed directly and compared with existing public databases for identification and quantification. Bioaerosol samples are first eluted from the collection device and concentrated; DNA material is then extracted and purified for subsequent PCR analysis. The PCR method detects both culturable and nonculturable microorganisms, thus circumventing the limitations imposed by culturedbased techniques. Indeed, this method can be applied to any biological matter that contains nucleic acids and has been successfully applied to bioaerosols collected through a variety of sampling techniques (Peccia and Hernandez, 2006) . Modern PCR-based methods also provide results more rapidly than culturing techniques-on the order of minutes as compared to days (Wei et al., 2015) . The rapid developments in this technology have made it even more accurate, sensitive and also readily available to many laboratories. Because this method does not differentiate nucleic acid material from dead versus living microorganisms or cell debris, PCR results tend to overestimate the infectivity of bioaerosols.

Live unculturable microorganisms and dead but morphologically intact microorganisms can be also quantified by fluorescence-based methods. Non-culturable bioaerosols collected using air filtration or liquid impinger methods are transferred to the lab. There the microorganisms can be stained with a fluorophore (e.g. acridine orangesyto9, DAPI) that binds with nucleic acid, and counted with an epifluorescence microscope or flow cytometry. Using a combination of fluorescent dyes such as the live/dead BacLight assay, both live and dead microorganisms can be visualized and enumerated. Specific identification and quantification of a certain bacteria species is also possible with the use of fluorescentlylabeled nucleic acid probes to target rRNA within morphologically intact cells. The main advantage of these methods is that all microorganisms (i.e. culturable and unculturable, dead and living) are quantified.

The estimated bioaerosol concentration is therefore often higher than values determined using culturebased methods (Pepper et al., 2015) .

Apart from whole microorganisms, bioaerosols also contain toxic or allergenic microbial byproducts that cannot be quantified by nucleic acid-based methods. The most common method for the detection of this J o u r n a l P r e -p r o o f Journal Pre-proof type of bioaerosol is enzyme-linked immunoassays (ELISA). Toxin/allergen bioaerosol samples are first immobilized to a solid surface, a detection antibody which recognizes a specific antigen on the toxin/allergen is then added. The detection antibody can be either directly linked to an enzyme or detected by a secondary antibody which is linked to an enzyme. When a substrate of the enzyme is added to the mixture, it produces a signal (e.g. chemiluminescence, fluorescence, absorbance) that correlates with the quantity of the antigen in the bioaerosol sample. ELISA assays have also been developed to detect and quantify microbial pathogens by detecting the antigens on the microbial surface. Successful applications include detection of bacterial spores (Stratis-Cullum et al., 2003) , E.coli (Korzeniewska and Harnisz, 2012) , and mite allergen (Miyajima et al., 2014) . The advantages of ELISA are its specificity and adaptability to direct field use; the disadvantage is that a good antibody is often required, which is not always available.

3.1. The aeration basin. The aeration basin is a central feature in modern wastewater treatment process trains. The purpose of the aeration basin is to remove biodegradable wastewater constituents, primarily organic compounds and nitrogen. Large populations of prokaryotic and eukaryotic species are cultivated in the aeration basin by controlling pH, proper mixing, and providing oxygen as a terminal electron acceptor. Aeration is achieved with diffused aeration or mechanical mixing (e.g. surface aeration). The intense mixing and turbulence creates bioaerosol emission. Numerous studies have documented bioaerosol emission from aeration basins (Bauer et al., 2002; Brandi et al., 2000; Filipkowska et al., 2000; Han et al., 2019) .

Previous studies have used culture-dependent and cultureindependent methods to identify the microorganisms present in bioaerosols emitted from aeration basins (Table 1 ). The primary focus of the culture-dependent work was the identification of known clinical pathogens including intestinal microorganisms (e.g. Enterococci sp., Enterobacter sp.) and bacteria that J o u r n a l P r e -p r o o f Journal Pre-proof inhabit mucous membranes (e.g. Staphylococcus sp.). Therefore, exposure to bioaerosols emitted from aeration basins create human health risks. Korzeniewska et al., 2009 also detected fungi, which are resilient in bioaerosols because of their ability to survive desiccation stress (Pepper et al., 2015) . The culture-independent bioaerosol work of Gaviria-Figueroa et al., 2019 revealed the presence of a variety of microorganisms that have functional significance in the wastewater treatment process. For example, Candidatus Accumulibacter is responsible for phosphorus removal (Metcalf and Eddy, 2003) . Nitrospira is responsible for the oxidation of nitrite, a necessary step in the nitrification process (Metcalf and Eddy, 2003) . Gaviria-Figueroa et al., 2019 also detected antibiotic resistance gene-carrying organisms in bioaerosols.

Prolonged viability is a concern for bioaerosols released from aeration basins. Activated sludge microorganisms grow within flocculent aggregates, consisting of extracellular polymeric substances (EPS), inert particles, water, and numerous ionized chemicals (Metcalf and Eddy, 2003 The bubble then bursts when the membrane can no longer maintain the internal pressure of the bubble ( Figure 1A) . As a bubble dissolves, the liquid remaining in the membrane splits into strands of liquid that J o u r n a l P r e -p r o o f Journal Pre-proof then break into small droplets a few microseconds later ( Figure 1B ). If the bubble is only a few millimeters in diameter, a jet may form as water rushes to fill the void space created by the bubble on the surface ( Figure 1C ). However, in larger bubbles, there is some confusion about the conditions that form jets; Ke et al. (2017) indicated that this jet does not form above a diameter of 3.4 mm, so that the only source of aerosols is from the droplets formed from the membrane during bursting. Lee et al. (2011) described jetting in the bursting of bubbles with a radius length much greater than 100 µm, but it is not quite clear whether those larger bubbles were as large as the ones examined in Ke et al. (2017) . Bursting bubbles release pollutants and ions into the atmosphere (Hardy, 1982) . Microorganisms accumulate in the interface that separates the bulk liquid from the atmosphere (Hermansson and Dahlbäck 1983; Schäfer et al., 1998) . Numerous studies have shown that bursting bubbles aerosolize the microorganisms present in the air-water interface (Aller et al., 2005; Baylor et al., 1977, Blanchard and Syzdek, 1970; Filipkowska et al., 2000) . Larger bubbles emit more and larger aerosol particles than smaller ones (Ke et al., 2017) .

with horizontal rotating brushes (Figure 2 ). Droplets of various sizes are ejected from the water into the air off of the discs in all directions ((1) in Figure 2 ). Many of these droplets may be too large to become aerosols and will simply fall back into the water. However, droplets < 7 µm in diameter can be carried on moving air currents and are small enough to be deposited in the respiratory system during normal respiration (Donnison et al., 2004) . Foams and bubbles are commonly observed near the rotating discs on the water surface ((2) in Figure 2 ). Bioaerosols are released as these bubbles pop on the surface and by the splashing that occurs when droplets land on the water surface ((3) in Figure 2 , Fracchia et al., 2006; Li et al., 2013; Sánchez-Monedero et al., 2008) . Bubbles form from the splashing of water caused by the rotation of the brushes ((4) in Figure 2 ) and from the forcing of air down into the water column.

Splashing and bubble bursting occur with other methods of mechanical surface aeration (e.g. subsurface turbines, fountains, horizontal paddles), but peer-reviewed studies have not yet revealed the bioaerosol release mechanisms for these processes. Horizontal mixing rotors were found to produce higher aerosol 

4.1 Covering sources. Aeration basins can be covered with light-weight materials which can be periodically cleaned or replaced. This approach is now commonly done for odor control (Metcalf and Eddy, 2003) . Hinged lids have been used to cover bioaerosol sources at a full-scale treatment facility (Fernando and Fedorak, 2005) . Other studies have recommended covering sources to reduce bioaerosol emissions (Fathi et al., 2017; Kummer and Thiel, 2008) or designing facilities so that sewage is not exposed to open air (O'Hara and Rubin, 2005) .

The results in Fernando and Fedorak (2005) indicated that a floating cover apparently does not affect oxygen transfer for diffused aeration, but there may be a marginal reduction in oxygen transfer efficiency and energy efficiency (Ashley et al., 1992) . Covers may also have small openings to allow for airflow out of the basin during aeration (Fernando and Fedorak, 2005) , which may permit bioaerosol release.

However, these hinged coverings may not be practical for certain mechanically-aerated systems. Since the mechanical aeration methods are designed to introduce open air into the wastewater, oxygen flow may be restricted when the basin is covered, resulting in a drop in the aeration efficiency of the rotors.

Bioaerosol emissions from activated sludge basins are dependent upon the aeration method, which in turn influences mixing. Switching from surface aeration to fine bubble diffused aeration appears to reduce bioaerosol production (Bauer et al., 2002; Brandi et al., 2000; Fathi et al., 2017; Han et al., 2019; Sánchez-Monedero, 2008) . Fernando and Fedorak, 2005 showed that changing from coarse bubble to fine bubble aeration decreased the bioaerosol concentrations from 1000 and 1800 CFU/m 3 to 24 and 37 CFU/m 3 and two locations near the aeration basins.

Changing from coarse bubble aeration to fine bubble aeration is a promising strategy for attenuating bioaerosol release. This option also has the additional benefit of improving oxygen transfer efficiency J o u r n a l P r e -p r o o f Journal Pre-proof (Metcalf and Eddy, 2003) . However, the conversion may require a redesign of the apparatus delivering the air to the basins. Characteristics of the bioaerosols produced by these methods must also be considered. Although horizontal rotors exert stronger shear forces that produce more total bioaerosols, the size of those particles is predominantly greater than 7.0 µm (Han et al., 2020) . Meanwhile, fine bubble aeration mainly produces smaller, respirable aerosols (Han et al., 2020) .

Another strategy is the use of free-floating carrier media (FFCM), which consists of low-density materials that could be placed on the water surface to stop bioaerosol release. Bourke, 1999 and Sheehy et al., 1984 described using plastic or polystyrene beads as FFCM to prevent mists from being released from electroplating tanks. Shredded foam has also been used because it may interlock better than sphere-shaped media (Bourke, 1999). Hung et al. (2010) used polystyrene spheres in a laboratory-scale bioreactor, and they found that the emission reduction increased as the sphere size (i.e. 1.9, 2.9, 3.4, 4.8 cm) decreased. They also showed that using multiple bead layers may provide a modest improvement in capture efficiency. For both the electroplating and sewage aeration experiments, free floating carrier media was effective at removing or preventing the release of most aerosol particles up to around 90% (Hung et al., 2010; Sheehy et al., 1984) . Noh et al., 2019 found that the introduction of powdered activated carbon (PAC) in a membrane bioreactor increased the formation of bacterial flocs and decreased the amount of free bacteria available to be aerosolized. No FFCM studies have been carried out at a full-scale wastewater treatment plant to the best knowledge of the authors.

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P r e -p r o o f Reference Identification (i.e. order, family, genus, or species) or description Method Brandi et al., 2000* Enterococci sp., Escherichia coli, staphylococci sp.; total bacteria Culture-selective media

* Shigella spp., Yersinia enterocolitica, Escherichia coli, Klebsiella pneumoniae ozaenae, Staphylococcus sp., Pseudomonas sp., actinomycetes sp

Enterobacter sp; Bacillus sp., Acinetobacter sp., Lysinibacillus Culture-selective media

Clostridium perfringens, Staphylococcus aureus, Enterococcus spp. Culture-selective media

Salmonella enteritidis, Salmonella boydii, reovirus, enterovirus Culture-selective media

Alphaproteobacteria: Rhodobacter

Nitrospirae: Nitrospira 16S rRNA gene sequencing and cultureselective media

* Mesophilic bacteria, TSA-SB bacteria Culture-selective media

Chryseobacterium sp., Stenotrophomonas sp., Alcaligenes sp., Micrococcus sp., Pantoea sp., Enterobacter sp

** Salmonella sp., total viable bacteria Culture-selective media and PCR

Enterobacteriaceae sp