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

Appl Environ Microbiol. 2015 Aug;81(15):5134-43. doi: 10.1128/AEM.00998-15. Epub 2015 May 22.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacteria / classification*
  • Bacteria / genetics
  • Bacteria / isolation & purification*
  • Bacteriological Techniques / methods*
  • DNA Fingerprinting / methods*
  • DNA, Bacterial / chemistry
  • DNA, Bacterial / genetics
  • DNA, Ribosomal / chemistry
  • DNA, Ribosomal / genetics
  • Feces / microbiology*
  • High-Throughput Nucleotide Sequencing
  • RNA, Ribosomal, 16S / genetics
  • Sequence Analysis, DNA
  • Temperature
  • Wastewater / microbiology*
  • Water Purification*


  • DNA, Bacterial
  • DNA, Ribosomal
  • RNA, Ribosomal, 16S
  • Waste Water