Automated Sampling Procedures Supported by High Persistence of Bacterial Fecal Indicators and Bacteroidetes Genetic Microbial Source Tracking Markers in Municipal Wastewater during Short-Term Storage at 5°C

Abstract

e Because of high diurnal water quality fluctuations in raw municipal wastewater, the use of proportional autosampling over a period of 24 h at municipal wastewater treatment plants (WWTPs) to evaluate carbon, nitrogen, and phosphorus removal has become a standard in many countries. Microbial removal or load estimation at municipal WWTPs, however, is still based on manually recovered grab samples. The goal of this study was to establish basic knowledge regarding the persistence of standard bacterial fecal indicators and Bacteroidetes genetic microbial source tracking markers in municipal wastewater in order to evaluate their suitability for automated sampling, as the potential lack of persistence is the main argument against such procedures. Raw and secondary treated wastewater of municipal origin from representative and well-characterized biological WWTPs without disinfection (organic carbon and nutrient removal) was investigated in microcosm experiments at 5 and 21°C with a total storage time of 32 h (including a 24-h autosampling component and an 8-h postsampling phase). Vegetative Escherichia coli and enterococci, as well as Clostridium perfringens spores, were selected as indicators for cultivation-based standard enumeration. Molecular analysis focused on total (AllBac) and human-associated genetic Bacteroidetes (BacHum-UCD, HF183 TaqMan) markers by using quantitative PCR, as well as 16S rRNA gene-based next-generation sequencing. The microbial parameters showed high persistence in both raw and treated wastewater at 5°C under the storage conditions used. Surprisingly, and in contrast to results obtained with treated wastewater, persistence of the microbial markers in raw wastewater was also high at 21°C. On the basis of our results, 24-h autosampling procedures with 5°C storage conditions can be recommended for the investigation of fecal indicators or Bacteroidetes genetic markers at municipal WWTPs. Such autosampling procedures will contribute to better understanding and monitoring of municipal WWTPs as sources of fecal pollution in water resources. Microbial fecal contamination of aquatic systems by municipal wastewater represents a significant threat to public health (1). Thus, appropriate wastewater disposal technologies and fecal pollution monitoring programs are critical for safeguarding our water resources. Standard fecal indicators, as well as recently developed genetic microbial source tracking (MST) markers, are used to monitor the microbial fecal loads emitted from wastewater treatment plants (WWTPs) and their impact on receiving waters (2-6). Microbiological sampling of WWTPs is commonly based on manually recovered samples (7). However, the concept behind these methods neglects temporal fluctuations in water quality. Large diurnal variations have been reported for key chemical parameters, such as nutrients, in raw wastewater (8). Determination of the efficacy of carbon, nitrogen, and phosphorus removal at WWTPs is thus frequently based on automated diurnal sampling. For example, in Austria, auto...

Persistence of Pathogens in Sewage and Other Water Types

“…This was consistent with findings of the persistence of human specific Bacteroides (BacHum-UCD; AllBac) in raw wastewater and secondary effluent, where a 1 log 10 reduction was only observed at 5°C after 11 days (Mayer et al 2015). The same log reduction was observed at 4 days when the temperature was 21°C, further highlighting the importance of temperature on the survival of some bacteria in wastewater (Mayer et al 2015). In contrast, Mayer et al (2015) observed no significant difference in die off of Clostridium perfringens in raw wastewater and secondary effluent at cold (5°C) and warm (21°C) temperatures.…”

“…For example, at colder temperatures (5°C) Brooke et al (2015) observed a 1 log 10 die-off of Bacteroides thetaiotaomicron only after 9.6 days, however at temperatures ranging from 25 to 43°C, T90s of 1 to 1.8 were reported. This was consistent with findings of the persistence of human specific Bacteroides (BacHum-UCD; AllBac) in raw wastewater and secondary effluent, where a 1 log 10 reduction was only observed at 5°C after 11 days (Mayer et al 2015). The same log reduction was observed at 4 days when the temperature was 21°C, further highlighting the importance of temperature on the survival of some bacteria in wastewater (Mayer et al 2015).…”

“…The same log reduction was observed at 4 days when the temperature was 21°C, further highlighting the importance of temperature on the survival of some bacteria in wastewater (Mayer et al 2015). In contrast, Mayer et al (2015) observed no significant difference in die off of Clostridium perfringens in raw wastewater and secondary effluent at cold (5°C) and warm (21°C) temperatures. Yeager and Ward (1981) examined the survival of pathogens in sewage sludge at 21°C and found that in liquid sludge (5% solids) the pathogens Salmonella typhimurium, Enterobacter spp.…”

“…It seems to be most persistent at low temperatures (2 to 6°C), where it is shown to survive for >11 days before seeing a 1 log 10 reduction. In secondary wastewater effluent, Mayer et al (2015) found E.coli spp. to be less persistent and observed a 1 log 10 die-off after 4 to 11 days.…”

Evaluation of Molecular Methods for the Detection and Quantification of Pathogen-Derived Nucleic Acids in Sediment

“…The CTAB-based extraction has been widely used for studying microbial communities in soil (Amos et al, 2015; Mayer et al, 2015; Santos et al, 2015), and this study supports its usefulness for the recovery of high abundance viruses and bacteria from sediment. It is a cost-effective approach and requires less preparation time than the PowerSoil® Kit (Table 1).…”