Source Bioaerosol Concentration and rRNA Gene-Based Identification of Microorganisms Aerosolized at a Flood Irrigation Wastewater Reuse Site

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 microorganisms, culture-based assays for heterotrophs and gram-negative enteric bacteria, and small-subunit rRNA gene-based cloning were used for microbial characterizations of aerosols and effluent wastewater samples. Bioaerosol results were divided into two wind speed regimens: (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 air-borne concentration of total microorganisms, culturable heterotrophs, and gramnegative enteric bacteria were, respectively, 1.1, 4.2, and 6.2 orders of magnitude greater during the highwind-speed regimen. Small-subunit rRNA gene clone libraries processed from 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.

Accuracy, Precision, and Method Detection Limits of Quantitative PCR for Airborne Bacteria and Fungi

“…DNA extraction methods used here were included because they are commonly used in environmental analysis and because many aerosol labs have limited expertise and resources in molecular biology and rely on kits for DNA extraction. More rigorous custom methods for extracting and recovering DNA from aerosols have been published (15,43) although direct comparisons with the DNA presented here are complicated by the fact that these literature values are based on comparative efficiencies rather than on absolute efficiencies. Beyond DNA, extraction efficiencies of whole cells from common aerosol filter material have not been systematically reported in the literature (16,24,40,44).…”

Source Tracking Aerosols Released from Land-Applied Class B Biosolids during High-Wind Events

“…DNA was then isolated by bead beating to lyse cells, phenol-chloroform extraction to separate DNA, and column filtration for DNA cleanup and concentration. This method followed a previously described method (30), with the exception that no lysozyme was added and the bead-beating rate was set at 2,500 rpm. The method described above was also used to isolate DNA from bulk soil and biosolid samples.…”

Qualitative and quantitative methodologies for determination of airborne microorganisms at concentrated animal-feeding operations

“…Filter materials that have been successfully used in PCR-based bioaerosol studies using liquid samples from glass impingers are Nytran (Alvarez et al 1994), polycarbonate (Paez-Rubio et al 2005), nylon (Alvarez et al 1995), and Teflon (Alvarez et al 1995). Aerosol samples can also be directly impinged onto filters for subsequent PCR analysis; filters used for this purpose are tracked-etched polyester (Wilson et al 2002a), polycarbonate (Zeng et al 2004), and polyethersulfone (Stärk et al 1998).…”

“…As an alternative to culture-based techniques, the detection of microorganisms in aerosols by PCR has become increasingly popular over the last two decades (Alvarez et al 1994;Wakefield 1996;Stärk et al 1998;Olsson et al 1998;Williams et al 2001;Wu et al 2003;Zeng et al 2004;Paez-Rubio et al 2005;An et al 2006) allowing for the detection of target nucleic acid sequences, thereby eliminating the need to cultivate microorganisms for their detection and identification. This is particularly useful for microorganisms that are difficult to culture, slow growing or have never been cultured before, providing increased sensitivity over traditional culture-based methods (Josephson et al 1993;Alvarez et al 1994).…”

“…Aerosol samples can also be directly impinged onto filters for subsequent PCR analysis; filters used for this purpose are tracked-etched polyester (Wilson et al 2002a), polycarbonate (Zeng et al 2004), and polyethersulfone (Stärk et al 1998). The filters are added to sterile distilled water (Alvarez et al 1995) or buffer solution (Wilson et al 2002a;Zeng et al 2004;Paez-Rubio et al 2005) and then the microorganisms are eluted via agitation such as vortexing, shaking, or sonication.…”