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SOURCE BIOAEROSOL CONCENTRATION AND rDNA-BASED IDENTIFICATION OF MICROORGANISMS AEROSOLIZED DURING AGRICULTURAL WASTEWATER REUSE

Tania Paez-Rubio* Arizona State University. Emily J. Viau Arizona State University. MC. Socorro Romero-Hernandez Universidad Autnoma de Baja California. Jordan Peccia Arizona State University. Author address: Arizona State University. Engineering Center G-wing. Room 252. Tempe. 85287-5306. Phone number: 001-480-965-3296. Fax number: 001-480-965-0557. e-mail: tania.paez@asu.edu ABSTRACT Reuse of partially treated domestic wastewater for agricultural irrigation is a growing practice in arid regions throughout the world. A field sampling campaign to determine bioaerosol concentration, culturability, and identity at various wind speeds was conducted at a flooded wastewater irrigation site in Mexicali, Baja California, Mexico. Direct fluorescent microscopy measurements for total airborne microorganisms, culture-based assays for heterotrophs and gram-negative enteric bacteria, and small subunit rRNA-based cloning were utilized for microbial characterizations of aerosols and effluent wastewater samples. Bioaerosol results were divided into two wind speed regimes: (i) below 1.9 m/s, average speed 0.5 m/s, and (ii) above 1.9 m/s, average speed 4.5 m/s. Average airborne concentration of total microorganisms, culturable heterotrophs, and gram-negative enteric bacteria were respectively 1.1, 4.3, and 5.3 orders of magnitude greater during the high wind speed regime. Small subunit rDNA encoding gene clone libraries based on samples from air and the irrigation effluent wastewater during a high wind sampling event indicate that the majority of air clone sequences were more than 98% similar to clone sequences retrieved from the effluent wastewater sample. Overall results indicate that wind is a potential aerosolization mechanism of viable wastewater microorganisms at flood irrigation sites. Keywords: wastewater reuse, aerosolization, bioaerosols, culturability, 16S rDNA library. INTRODUCTION In the arid border region of Mexico and the United States, a rapidly growing population, an agricultural-based economy, and the low availability of surface and ground water underscore the importance of water reuse (Gallegos et al., 1999). The use of effluent wastewater for agricultural irrigation is therefore a growing practice in this region. Throughout the world, an estimated 80% of domestic wastewater produced in developing countries maybe reused for irrigation (Cooper, 1991). The World Health Organization (WHO, 1989) has defined a coliform guideline for unrestricted irrigation using domestic wastewater, allowing 1,000 fecal coliforms per 100 ml. This standard is difficult to meet in many developing countries where the wastewater designated for crop irrigation most commonly receives partial treatment and is rarely disinfected (WHO, 2002). Among workers and occupants of surrounding communities, lack of disinfection and exceedance of indicator organism guidelines have prompted concerns regarding the elevated health risk caused by aerosolization and subsequent transport of effluent wastewater microorganisms (Bausum et al., 1982, Bausum et al., 1983, Sorber et al., 1976, Teltsch and Katzenelson, 1978). Rapid urbanization of many cities along the U.S.-Mexico border has led to the growth of populations near agricultural land and may exacerbate the risk. In addition to respiratory tract infections, it has been suggested that aerosolized enteric microorganisms common in wastewater may produce intestinal tract infections when particles are deposited in the upper nasal-pharynx and later swallowed (Sorber et al., 1975). Epidemiological studies (Katzenelson et al., 1976, Shuval et al., 1989) in agricultural communal settlements located near fields using wastewater irrigation indicated an increase in the rate of enteric diseases during the irrigation season. This increase was most evident in the youngest population group (0-5 years). Negative health impacts linked to wastewater bioaerosols have also been

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documented in wastewater workers (Khuder et al., 1998), in which a positive correlation between bioaerosol exposure and gastrointestinal infections has been observed. Moreover, workers exposed to airborne gram-negative enteric bacteria and endotoxins at industrial and municipal wastewater treatment plants reported health issues such as respiratory symptoms, fever, and tiredness (Laitinen et al., 1994, Melbostad et al., 1994). Previous wastewater bioaerosol studies have measured above ambient airborne bacterial concentrations near wastewater systems that have an overt potential for aerosolization. These systems include open activated sludge basins (Brandi et al., 2000, Fannin et al., 1985, Laitinen et al.,1994), and spray irrigation sites (Bausum et al., 1983, Bitton, 1999, Shuval et al., 1989, Teltsch and Katzenelson, 1978, Teltsch et al., 1980). Although wastewater flood irrigation is the more common practice, the effects of wind velocity on microorganism aerosolization have not been characterized. Fundamental information to determine the relationship between bioaerosol concentration, identity, and environmental conditions such as wind velocity is needed to assess and predict human exposure. In the present study, a field sampling campaign to characterize bioaerosols collected during periods of both low and high wind speeds and dial solar conditions was conducted at a flooded wastewater irrigation site in Northern Mexico. Directly fluorescent microscopy measurements for total airborne bacteria and culture-based assays were applied to investigate the effect of environmental conditions on the total concentration and culturability of bioaerosols. Phylogenetic identification and comparison of the aerosol and effluent wastewater microbial communities were made by construction of clone libraries based on the sequence analysis of the SSU rDNA-encoding gene. METHODOLOGY AND DEVELOPED STAGES Aerosol and effluent wastewater sampling. Studies were conducted at an agricultural field located in the Northern Sonoran Desert on the urban fringe of Mexicali, Baja California, Mexico. The field was seeded for Bermuda grass sod and flood irrigated with undisinfected effluent from a two stage anaerobic-facultative lagoon that treated domestic wastewater. The fecal coliform concentration in the effluent wastewater is commonly more than 103 CFU/ml (CEPA, 2002) and a strong H2S odor was present near the effluent wastewater. Effluent wastewater for flood irrigation was applied 12 hours prior to sampling, and wastewater application continued, ensuring wastewater presence, during the 3 day sampling campaign performed in November of 2002. Aerosol and effluent wastewater samples were taken five times daily: 7 am, 11 am, 2 pm, 4:30 pm, and midnight. Sampling equipment was located in the east side of the field and samplers were directed toward the field. A weather station, Weather Monitor II (Davis Instrument Corporation, Hayward, CA) was set at 1.5 m height near the sampling site and provided relative humidity, wind speed and direction, and temperature measurements. Solar radiation was measured with a radiometer/photometer, model IL1700 (International light, Newburyport, MA) with SED005#776 and WBS320#24343, detector and filter specifications, respectively. This filter has a response in the 250-400 nm range (UV-B and UV-A). All meteorological data were recorded every five minutes during each sampling event. For aerosol sampling, three sterile glass liquid impingers (Biosamplers, SKC Inc., Eighty four, PA) were used simultaneously. Impingers were connected to a HP vacuum pump (GAST, Benton Harbor, MI) through a galvanized iron manifold and suspended at the breathing zone height, approximately 1.5 m above the ground. The pump operated at a pressure drop greater that 0.5 atm., and the flow rate in each impinger was 12.5 0.5 L/min. Cells were collected into 15 ml of sterile phosphate buffered saline (PBS) (30 mM phosphate buffer, pH=7.2; 125mM NaCl) for 30 minutes. During sampling, impingers were wrapped with aluminum foil to avoid sunlight inactivation of collected cells. Aerosol samples for the phylogenetic library were collected using a MicroVIC bioaerosol concentrator (Mesosystems Technology, Kennewick, WA). The MicroVIC collects particles below 10 m at a flow rate of 400 L/min and concentrates the sample into a flow of 12.5 L/min for liquid impingement. MicroVIC samplers were operated for 30 minutes. During each aerosol sampling time, effluent wastewater samples were taken from the irrigated site at a 5 cm depth. Upon collection all samples were immediately stored at 4oC in the dark (Li and Lin, 2001). Bacterial culturing and enumeration. Aerosol and effluent wastewater samples were diluted in PBS, and 0.2 ml of the sample was spread onto media plates in triplicate in accordance with standard methods (APHA, 1995). R2A agar media (Difco Laboratories, Detroit, MI) was used to produce heterotrophic plate counts (HPCs). Plates were incubated aerobically at room temperature for 5 days. MacConkeys agar (Difco Laboratories) was used for the select detection of culturable gram-negative enteric bacteria. MacConkey plates were incubated at 37C for 24 hours. Epifluorescent microscopy was used to enumerate total bacteria (culturable and nonculturable) in accordance with the previously described method for liquid impinged cells (Hernandez et al. 1999)). Cells were stained with 46-diamidino-2-phenylindole (DAPI) (Pierce, Rockford, IL) at a final concentration of 20 g DAPI /ml, and vortexed for 1 minute. Samples were then filtered onto a 25 mm diameter, 0.2 m pore-size, black polycarbonate membrane (Osmonics, Inc., Minnetonka, MN) and observed on an Olympus BX51/BX52 microscope (Olympus, Melville, NY) at 1000x magnification. All direct counts were

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produced by counting a minimum of eight random fields and more than 400 total cells on each filter. Samples with a coefficient of variance between fields greater than 20% were reanalyzed. In all aerosol culturable and direct counts, concentrations presented are averages of 3 separate aerosol samplers. Protocols for DNA extraction, cloning, and sequencing are described in detail in Paez-Rubio et al. (2004). RESULTS Aerosol microbial populations. Bioaerosol results were divided into two wind speed regimes: (i) below 1.9 m/s, average speed 0.5 m/s, and (ii) above 1.9 m/s, average speed 4.5 m/s. The concentrations of total and culturable airborne microorganisms obtained during the field sampling campaign are presented in Figure 1. Wind speed and wind direction are also included in this figure. No statistical difference was found between total and culturable concentrations for the last four samples (p 0.05). The average concentrations of total bacteria, heterotrophs (HPC), culturable gram-negative enteric bacteria, were 1.1, 4.3, and 5.3 orders of magnitude greater in samples collected during the high wind speed regime than those obtained during low wind speed. A tstudent analysis confirmed the aerosol concentration increase (p 0.05 for total bacteria and HPC, and p 0.17 for gram-negative enteric bacteria). Daytime and nighttime total and culturable aerosol concentrations were not different (p 0.05) when tested using all of the sampling data or a subset of data controlled for similar wind conditions. All aerosol measurements were taken under clear conditions. The average daily peak in solar radiation (UVA and UVB) was 2.1 1.0 (std. dev.) Mw*m-2, while minimum nighttime solar radiation averaged 3 x 10-6 1 x 10-6(std. dev.) mW*m-2. During the sample campaign relative humidity levels were stable, averaging 37% 9 %(std. dev.) and ranging from 21% to 51%. Figure 1: Average total and culturable airborne microbial concentrations adjacent to irrigated field at a height of 1.5 m. Bars: , gram-negative enteric bacteria; , Heterotrphic Plate Count (HPC); and , total bacteria. Wind speed is denoted by a dash ( ) and wind direction is listed on the x axis. Error bars denote standard deviation from three separate aerosol samples. Effluent wastewater microbial population. Prior to treatment, the general effluent wastewater fecal coliform concentrations for this lagoon are between 106 and 108 CFU/ml, and the biochemical oxygen demand is approximately 500 mg O2/L. Approximately 80% of biochemical oxygen demand and a 2-4 log removal of fecal coliforms are usually accomplished during the lagoon treatment (unpublished data). Total microorganisms, HPC, and gram-negative enteric

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bacteria concentrations for the effluent wastewater did not change significantly (p 0.05) throughout the sampling campaign. For all samples, the average concentration and standard deviation of total microorganisms, HPC, and gram-negative enteric bacteria were 1.6 x 108 1.3 x107 number/ml, 2.9 x 107 4.2 x106 CFU/ml, and 2.3 107 4.2 105,CFU/ml, respectively. Figure 2 presents a comparison between the ratio of HPC to total microorganisms for each effluent wastewater and the aerosol sample, and demonstrates that changes in the culturability of aerosol samples cannot be attributed to variations in the effluent wastewater culturability. FIGURE 2: Ratios HPC/total number of microorganisms in aerosol and effluent wastewater. Bars: , aerosol ratio; , effluent wastewater ratio. 11/08 2 pm. ratio value for the effluent wastewater sample was not recorded. Error bars denote standard deviation for the ratio of three separate aerosol and three separate wastewater samples. Phylogenetic analyses of air and wastewater populations. Aerosol and effluent wastewater populations were further characterized by building phylogenetic clone libraries based on SSU rDNA encoding gene sequences. Libraries were constructed from genomic DNA extracted from the final aerosol and effluent wastewater samples. These samples were chosen based on the high total microorganism, HPC, and gram-negative enteric bacteria aerosol concentrations, as well as a high average wind velocity (5.2 m/s). Several similarities between the aerosol (Air) and effluent wastewater (EWW) populations exist as depicted in the Eukaryotic phylogenetic tree (Fig. 3), and the Bacterial phylogenetic tree (Fig. 4). Clone sequences derived from both aerosol and effluent wastewater samples were present in each of these groups, and the Heteromita sp. and Spumella sp. both contained clones from aerosols and effluent wastewater that were more than 98% similar. Other clones present in aerosol samples were more than 97% similar to specific genera of the Proteobacteria that are commonly identified in domestic wastewater and are culturable on MacConkey agar. These genera include Aquaspirillum, Escherichia, Alcaligenes, Shewanella, Pseudomonas, and Pantoea. DISCUSSION Results obtained during this study suggest that viable wastewater microorganisms can be aerosolized from flood irrigation sites. This assertion is supported by data derived from the culturable and molecular analysis of effluent wastewater and aerosol samples. Changes in airborne concentrations, mainly culturable concentrations (four last points in Fig. 1), were analyzed by two approaches. First, from Fig. 2 we could assess the independency of culturability in air from wastewater, in which higher culturability in air was not correlated with higher culturability in wastewater (concentrations remained constant ( = 0.05) along the sample campaign). Comparable results have been presented in the literature (Bausum et al.

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FIG 3. Eukaryotic phylogenetic tree of 18S rDNA sequences obtained from the air and effluent wastewater and related sequences retrieved from GenBank. Closed circles at nodes indicate > 75% bootstrap support, half shaded circles represent 50% to 75% bootstrap support, and open circles represent less than 50% support from 1,000 resamplings. 1982, Fannin et al., 1985, Teltsch and Katzenelson, 1978). The second approach was to correlate changes in culturability with atmospheric parameters. No positive relationship was observed between culturable aerosol concentrations or culturability ratios and solar radiation levels. This lack of correlation was observed through statistical analysis using either all of the sampling data or analysis restricted to only high or low wind velocity regimes. Pilot-scale solar radiation studies on airborne E. coli have demonstrated that at the near 50% relative humidity, levels observed during the sampling campaign, airborne inactivation rates in this organism are mostly independent of solar radiation, as inactivation is dominated by nonsolar effects (Paez-Rubio and Peccia, 2004). However, statistical evidences to correlate an increase in culturability with changes in wind speed were found. At air sampling sites downwind and adjacent to the flooded fields, total, HPC, and indicator gram-negative enteric microorganism concentrations increased from low to high wind speed regime. In all cases, for the high wind speed regime (average velocity >1.9 m/s), samplers were oriented toward the irrigation field and directly into the wind. During the low wind speed regime (average velocity < 1.9 m/s), samplers faced toward the irrigated field and both into and away from the wind. In this regime, however, aerosol concentrations varied by less than one order of magnitude regardless of the wind direction (Fig. 1). In one of the few outdoor aerosol studies that measured total and culturable aerosols, Tong and Lighthart (1999) similarly detected an increase in the culturable fraction of cells (1:222 to 1:27, HPC to total) when sampling aerosols above a grass field in which the higher concentrations coincided with highest wind speeds. Therefore, higher wind conditions contributed to a greater aerosolization and shorter time for inactivation occurred. Analysis of the SSU rDNA clone library contained valuable information for understanding the aerosolization of wastewater microorganisms under high wind conditions. Greater than 50% of the clones in the aerosol library were more than 97% similar to either clones found in effluent wastewater or to Proteobacteria commonly found in domestic wastewater. Changes in the physical mixing height of the atmosphere, wind velocity, wind derived aerosol sources, airborne inactivation, and low culturability of environmental bacteria necessitate a combination of both culture-based and molecular-based techniques to characterize environmental sources of bioaerosols. These analyses applied to a sample where both low and high wind speeds were observed indicate that wind may be a substantial mode of aerosolization of viable wastewater microorganisms at flood irrigation sites. While other studies have also linked changes in culturable aerosol concentration near wastewater spray irrigation sites or activated sludge basins with changes in atmospheric stability (Bausum et al., 1983, Fannin et al., 1985, Teltsch and Katzenelson, 1978), this work describes the potential of wind as an aerosolization mechanism of culturable microorganisms contained in standing water. Given the drive to reuse

Spumella elongata, AJ236859

0.1

EWW U44, AY534165 Unidentified rhodophyte PRD01a010B, AF289158 Air U89, AY532461

Uncultured Cercozoan LEMD080, AF372737 Heteromita globosa, U42447

Air U80, AY532453 EWW U78, AY534182 EWW U58, AY534170

EWW U61, AY534173

Spumella danica, AJ236861 EWW U59. AY534171

Spumella sp. 15G, AJ236857 Air U13, AY532426 Air U119, AY532481

Air U117, AY532479 EWW U56, AY534168

Air U44, AY532436 Air U50, AY532438 EWW U54, AY534167

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effluent wastewater for irrigation in water scarce regions throughout the world, the occurrence of high wind events poses an increased potential of human exposure in workers and nearby residents through the airborne route.

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0.1

Air U83, AY532456 EWW U101, AY534189

Flavobacterium aquatile, M62797 EWW U08,AY551561

Air U95, AY532465 Flavobacteriales bacterium CF1, AY145539

Air U115, AY532480 EWW U01, AY551560

Gelidibacter sp. 4-3, AF513398 Air U12, AY532425Flexibacteraceae bacterium, AY264840

Air U104, AY532472Bacteroidetes bacterium, AY133105

Air U85,AY532457 EWW U70, AY551563EWW U49, 551562

EWW U53, AY534166Rhizobium galegae, D12793

EWW U73, AY534179Candidatus Devosia euplotis, AJ548825

EWW U77, AY534181 Air U15, AY532427

EWW U103, AY534191Desulfomonile limimaris, AF230531Desulfomonile sp., AJ316022

Air U107, AY532473Air U72, AY532450Air U42, AY532434

Air U66, AY532444 Air U96, AY532466

Air U35, AY532429Exiguobacterium acetylicum, D55730Exiguobacterium undae, AJ344151

Air U54, AY532440 Bacillus benzoevorans, X60611

Air U109, AY532475Air U02, AY532421 Air U102, AY532470

Sludge bacterium A28, AF234746 Clostridium sticklandii, M26494Acholeplasma palmae, L33734 Acholeplasma axanthum, AF412968 Uncultured bacterium B02r010, AY197395 Air U77, AY532451

Air U82R, AY532455Air U78, AY532452Air U70, AY532448

EWW U04, AY534154EWW U09, AY534155Air U47, AY532437Air UA33, AY532428

Pseudomonas syringae, Z76669 Air U11, AY532424

Air U98, AY532468Pseudomonas stutzeri, AJ312165

EWW U41, AY534163EWW U69, AY534168Pseudomonas anguilliseptica, X99540

EWW U36, AY534160Pantoea agglomerans GSPB450, AF373197 Pantoea stewartii LMG2715, Z96080

Air U93, AY532464Air U67, AY532445

Air U86, AY532458Air U99, AY532469

Escherichia coli, Z83205Shigella flexneri, X96963

Aeromonas veronii, AF418213EWW U108, AY534192

Shewanella putrefaciens, X81623EWW U35, AY534159

Air U87, 532459 EWW U05, AY534153

Air U81, AY532454Alcaligenes sp. LMG5906, AY13121

Air U97, AY532467EWW U39, AY534262

Aquaspirillum serpens, AB074518EWW U42, AY534164

EWW U60, AY534172EWW U66, AY534177EWW U03, AY534152

Azoarcus communis, AF011343 EWW U80, AY534183

Dechlorimonas sp., AJ318917Air U114, AY532478

EWW U63, AY534175EWW U102, AY534190

Hydrogenophaga pseudoflava, AJ420327Air U06, AY532423 EWW U33, AY534158EWW U65, AY534176

Aquaspirillum metamorphum, Y18618Air U108, AY532474

Aquaspirillum delicatum, AF078756Acidovorax sp., AY177768

EWW U57, AY534169EWW U29, AY534180

Comamonadaceae bacterium BP-1b, AY145570 Air U113, AY532477

EWW U22, AY534157

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FIGURE 4: Prokaryotic phylogenetic tree of 16S rDNA sequences obtained from the air and effluent wastewater and related sequences retrieved from GenBank. Closed circles at nodes indicate > 75% bootstrap support, half shaded circles represent 50% to 75% bootstrap, and open circles represent less than 50% support from 1,000 resamplings. REFERENCES

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Bausum, H., Schaub, S., Bates, R., McKim, H., Schumacher, P., and B. Brockett. (1983) Microbial aerosols from a field-source wastewater irrigation site system. Journal WPCF. vol. 55, 65-75.

Bitton, G. (1999). Wastewater microbiology. John Wiley and Sons, New York. Brandi, G., Sisti, M., and G. Amagliani. (2000) Evaluation of the environmental impact of the microbial aerosols

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