Water, Air, & Soil Pollution: Focus

, Volume 8, Issue 1, pp 107–115 | Cite as

Determination of Enterobacteria in Air and Wastewater Samples from a Wastewater Treatment Plant by Epi-Fluorescence Microscopy

  • N. Patentalakis
  • A. Pantidou
  • N. KalogerakisEmail author


Bacteria, fungi and viruses are often encountered in aerosols and they can be pathogenic or cause allergies following inhalation. Wastewater treatment facilities have been found to generate bioaerosols, which are transported by the prevailing winds downstream to areas that can be up to several hundred meters away. Bioaerosol formation has a significant effect on air quality in the vicinity of the treatment plants. The amount and characteristics of the formed bioaerosols depend on the aeration system employed at the aeration tank of the wastewater treatment facility. In this work we determined Enterobacteria in air and wastewater samples at the main wastewater treatment facility of the city of Chania (Crete, Greece). Concentrations of airborne bacteria were measured near the aeration and sedimentation tanks. Samples of airborne bacteria were taken by using Merck’s MAS-100 bioaerosol collector followed by incubation and enumeration of the colonies. The use of different growth media enabled the separation and enumeration of several classes of microorganisms. As part of this study, Enterobacteria in air samples were also determined by filtration sampling followed by analysis of the collected microorganisms using DAPI staining to determine total cell counts (both viable and non-viable cells). Fluorescence in situ hybridization (FISH) with specific 23S rRNA probes was also used in order to identify specific groups of microorganisms (well known pathogens) present in the bioaerosols. The analysis was also performed in wastewater taken from the aeration and secondary sedimentation tanks in an effort to correlate the airborne bacteria with those in the wastewater.


Escherichia coli Klebsiella pneumoniae DAPI FISH Wastewater treatment plant Bioaerosol 


  1. Bascom, R. (1996). Environmental factors and respiratory hypersensitivity: The Americas. Toxicology Letters, 86, 115–130.CrossRefGoogle Scholar
  2. Bauer, H., Fuerhacker, M., Zibuschka, F., Schmid, H., & Puxbaum, H. (2002). Bacteria and fungi in aerosols generated by two different types of wastewater treatment plants. Water Research, 36, 3965–70.CrossRefGoogle Scholar
  3. Brandi, G., Sisti, M., & Amagliani, G. (2000). Evaluation of the environmental impact of microbial aerosols generated by wastewater treatment plants utilizing different aeration systems. Journal of Applied Microbiology, 88, 845–852.CrossRefGoogle Scholar
  4. Clark, S. C. (1987). Health effects associated with wastewater treatment, disposal, and reuse. J. Water Pollution Control Federation, 59, 436–440.Google Scholar
  5. Cox, C. S., & Wathes, C. M. (1995). Bioaerosols handbook. New York, NY: Lewis.Google Scholar
  6. Griffiths, W. D., & DeCosemo, G. A. L. (1994). The assessment of bioaerosols: A critical review. Journal of Aerosol Science, 25, 1425–1458.CrossRefGoogle Scholar
  7. Carducci, A., Gemelli, C., Cantiani, L., Casini, B., & Rovini, E. (1999). Assessment of microbial parameters as indicators of viral contamination of aerosol from urban sewage treatment plants. Letters in Applied Microbiology, 28, 207–210.CrossRefGoogle Scholar
  8. Carducci, A., Tozzi, E., Rubulotta, E., Casini, B., Cantiani, L. Rovini, E., et al. (2000). Assessing airborne biological hazard from urban wastewater treatment. Water Research, 34, 1173–1178.CrossRefGoogle Scholar
  9. Douwes, J., Thorne, P., Pearce, N., & Heederik, D. (2003). Bioaerosol health effects and exposure assessment: Progress and prospects. Annals of Occupational Hygiene, 47, 187–200.CrossRefGoogle Scholar
  10. Fannin, K. F., Vana, S. C., & Jakubowski, W. (1985). Effect of an activated sludge wastewater treatment plant on ambient air densities of aerosols containing bacteria and viruses. Applied and Environmental Microbiology, 49, 1191–1196.Google Scholar
  11. Heng, B. H., Goh, K. T., Doraisingham S., & Quek, G. H. (1994). Prevalence of hepatitis A virus infection among sewage workers in Singapore. Epidemiology and Infection, 113, 121–128.CrossRefGoogle Scholar
  12. Jensen, P. A., & Schafer, M. P. (1998). Sampling and Characterization of bioaerosols. In: National Institute for Occupational Safety and Health (NIOSH) manual of analytical methods. Method 0800, pp. 82–112.Google Scholar
  13. Kalogerakis, N., Paschali, D., Lekaditis, V., Pantidou, A., Eleftheriadis, K., & Lazaridis, M. (2005). Indoor air quality – Bioaerosol measurements in domestic and office premises. Journal of Aerosol Science, 36, 751–761.CrossRefGoogle Scholar
  14. Lee, J. A., Johnson, J.C., Reynolds, S. J., Thorne, P. S., & O’shaughnessy, P. T. (2006). Indoor and outdoor air quality assessment of four wastewater treatment plants. Journal of Occupational and Environmental Hygiene, 3, 36–43.CrossRefGoogle Scholar
  15. Loy, A., Horn, M., & Wagner, M. (2003) probeBase – An online resource for rRNA-targeted oligonucleotide probes. Nucleic Acids Research, 31, 514–516.CrossRefGoogle Scholar
  16. Merck (1999). MAS-100 microbiological air sampler operator’s manual. Germany: Merck.Google Scholar
  17. Merck (2000). Microbiology manual. Germany: MerckGoogle Scholar
  18. Monn, Ch. (2001). Exposure assessment of air pollutants: A review on spatial heterogeneity and indoor/outdoor/personal exposure to suspended particulate matter, nitrogen dioxide and ozone. Atmospheric Environment, 35, 1–32.CrossRefGoogle Scholar
  19. Moter, A., & Gobel, U. B. (2000). Fluorescence in situ hybridization (FISH) for direct visualization of microorganisms. Journal of Microbiol Methods, 41, 85–112.CrossRefGoogle Scholar
  20. McCartney, H. A., Fitt, B. D. L., & Schmechel (1997). Sampling bioaerosols in plant pathology. Journal of Aerosol Science, 28, 349–364.CrossRefGoogle Scholar
  21. Pascual, L., Pérez-Luz, S., Yáñez, M.A., Santamaría, A., Gibert, K., Salgot, et al. (2003). Bioaerosol emission from wastewater treatment plants. Aerobiologia, 19, 261–270.CrossRefGoogle Scholar
  22. Pernthaler, J., Glöckner, F. O., Schönhuber, W., & Amann, R. (2001) Fluorescence in situ hybridization with rRNA-targeted oligonucleotide probes. In J. Paul (Ed.), Methods in microbiology: Marine microbiology (vol. 30, pp. 207–226). London, UK: Academic.Google Scholar
  23. Poulsen, O. M., Breum, N. O., Ebbehøj, N., Hansen, A. M., Ivens, U. I., Van Lelieveld, D., et al. (1995). Sorting and recycling of domestic waste. Review of occupational health problems and their possible causes. The Science of the Total Environment, 168, 33–56.CrossRefGoogle Scholar
  24. Prost, A., & Boutin, P. (1989). Sanitary risk resulting from the agricultural reuse of urban wastewater. Technical Science Methods, 84, 25–33.Google Scholar
  25. Ranalli, G., Principi, P., & Sorlini, C. (2000). Bacterial aerosol emission from wastewater treatment plants: Culture methods and bio-molecular tools, Aerobiologia, 16, 39–46.CrossRefGoogle Scholar
  26. Rylander, R. (1999). Health effects among workers in sewage treatment plants. Occupational and Environmental Medicine, 56, 354–357.Google Scholar
  27. Rylander, R., Andersson, K., Belin, L., Berglund, G., Bergstrom, R., Hanson, L. A., et al. (1976). Sewage worker’s syndrome. Lancet, 2(7983), 478–479.CrossRefGoogle Scholar
  28. Sawyer, B., Elenbogen, G., Rao, K. C. O’Brien, P., Zenz, D. R., & Lue-Hing, C. (1993). Bacterial aerosol emission rates from municipal wastewater aeration tanks. Applied and Environmental Microbiology, 59, 3183–3186.Google Scholar
  29. Thorn, J., & Kerekes, E. (2001). Health effects among employees in sewage treatment plants: A literature survey. American Journal of Industrial Medicine, 40, 170–179.CrossRefGoogle Scholar
  30. Thorne, P. S. (2000). Inhalation toxicology models of endotoxin- and bioaerosol-induced inflammation. Toxicology, 152, 13–23.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2007

Authors and Affiliations

  1. 1.Department of Environmental EngineeringTechnical University of CreteChaniaGreece

Personalised recommendations