Use of chemometric analyses to assess biological wastewater treatment plants by protozoa and metazoa monitoring

  • A. Luís AmaralEmail author
  • Cristiano S. Leal
  • A. Isabel Vaz
  • J. Carvalho Vieira
  • Andreia C. Quinteiro
  • M. Lourdes Costa
  • L. Miguel Castro


Protozoa and metazoa biota communities in biological wastewater treatment plants (WWTP) are known to be dependent of both the plant type (oxidation ditch, trickling filter, conventional activated sludge, among others) and the working operational conditions (incoming effluent characteristics, toxics presence, organic load, aeration, hydraulic and sludge retention times, nitrification occurrence, etc.). Thus, for analogous WWTP operating in equivalent operating conditions, similar protozoa and metazoa communities can be found. Indeed, the protozoa and metazoa biota monitoring can be considered a quite useful tool for assessing the functioning of biological WWTP. Furthermore, the use of chemometric techniques in WWTP monitoring is becoming widespread to enlighten interrelationships within the plant, especially when a large collection of data can be obtained. In the current study, the protozoa and metazoa communities of three different types of WWTP, comprising one oxidation ditch, four trickling filters, and three conventional activated sludge plants, were monitored. For that purpose, metazoa, as well as the main protozoa groups (flagellates, free-swimming, crawling and sessile ciliates, and testate amoeba) were determined in terms of contents and relative abundance. The collected data was further processed by chemometric techniques, such as cross-correlation, principal components, multivariate ANOVA, and decision trees analyses, allowing to successfully identify, and characterize, the different studied WWTP, and thus, being able to help monitoring and diagnosing operational problems.


Aerobic wastewater treatment plants Operational assessment Microbiota monitoring Principal component analysis Decision trees 


Funding information

This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684) and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by European Regional Development Fund under the scope of Norte2020—Programa Operacional Regional do Norte.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Avella, A. C., Görner, T., Yvon, J., Chappe, P., Guinot-Thomas, P., & de Donato, P. (2011). A combined approach for a better understanding of wastewater treatment plants operation: statistical analysis of monitoring database and sludge physico-chemical characterization. Water Research, 45(3), 981–992.CrossRefGoogle Scholar
  2. Bernat, K., Kulikowska, D., & Drzewicki, A. (2017). Microfauna community during pulp and paper wastewater treatment in a UNOX system. European Journal of Protistology, 58, 143–151.CrossRefGoogle Scholar
  3. Breiman, L., Friedman, J., Olshen, R., & Stone, C. (1984). Classification and regression trees. Belmont: Wadsworth.Google Scholar
  4. Canler, J. P., Perret, J. M., Duchéne, P., & Cotteux, E. (2011). Aide au diagnostic des stations d’épuration par l’óbservation microscopique dês boues actives. Versailles: Éditions Quae.Google Scholar
  5. Dubber, D., & Gray, N. F. (2009). Enumeration of protozoan ciliates in activated sludge: determination of replicate number using probability. Water Research, 43(34), 43–3452.Google Scholar
  6. Eikelboom, D. H. (2000). Process control of activated sludge plants by microscopic investigation. London: IWA Publishing.Google Scholar
  7. Einax, J. W., Zwanziger, H. W., & Geiss, S. (1997). Chemometrics in environmental analysis. VCH Verlagsgesellshaft: Weinheim.CrossRefGoogle Scholar
  8. Ganczarczyk, J. J. (1983). Activated sludge process: theory and practice. New York: Marcel Dekker.Google Scholar
  9. González, C. E., Salas, L. M., & Gutiérrez, A. C. (2016). A practical procedure for the microbiological monitoring of activated sludge plant functioning. Water Environment Journal, 30(3–4), 182–189.CrossRefGoogle Scholar
  10. Jenkins, D., Richard, M. G., & Daigger, G. T. (2003). Manual on the causes and control of activated sludge bulking, foaming, and other separation problems. London: IWA Publishing.Google Scholar
  11. Kinner, N. E., & Curds, C. R. (1987). Development of protozoan and metazoan communities in rotating biological contactor biofilms. Water Research, 21, 480–490.CrossRefGoogle Scholar
  12. Leal, C., Amaral, A. L., & Costa, M. L. (2016). Microbial-based evaluation of foaming events in full-scale wastewater treatment plants by microscopy survey and quantitative image analysis. Environmental Science and Pollution Research, 23, 15638–15650.CrossRefGoogle Scholar
  13. Liu, J., Yang, M., Qi, R., An, W., & Zhou, J. (2008). Comparative study of protozoan communities in full-scale MWTPs in Beijing related to treatment processes. Water Research, 42(8–9), 1907–1918.CrossRefGoogle Scholar
  14. Madoni, P. (1994). Estimates of ciliated biomass in activated sludge and biofilm. Bioresource Technology, 48, 245–249.CrossRefGoogle Scholar
  15. Madoni, P. (2011). Protozoa in wastewater treatment processes: a minireview. The Italian Journal of Zoology, 78(1), 3–11.CrossRefGoogle Scholar
  16. Madoni, P., & Ghetti, P. F. (1981). The structure of ciliated protozoa communities in biological sewage-treatment plants. Hydrobiologia, 83, 207–215.CrossRefGoogle Scholar
  17. Madoni, P., Davoli, D., & Chierici, E. (1993). Comparative analysis of the activated sludge microfauna in several sewage treatment works. Water Research, 27, 1485–1491.CrossRefGoogle Scholar
  18. Marín, I., Goñi, P., Lasheras, A. M., & Ormad, M. P. (2015). Efficiency of a Spanish wastewater treatment plant for removal potentially pathogens: characterization of bacteria and protozoa along water and sludge treatment lines. Ecological Engineering, 74, 28–32.CrossRefGoogle Scholar
  19. Martín-Cereceda, M., Pérez-Uz, B., Serrano, S., & Guinea, A. (2001). Dynamics of protozoan and metazoan communities in a full-scale wastewater treatment plant by rotating biological contactors. Microbiology Research, 156, 225–238.CrossRefGoogle Scholar
  20. Mesquita, D. P., Amaral, A. L., & Ferreira, E. C. (2011a). Identifying different types of bulking in an activated sludge system through quantitative image analysis. Chemosphere, 85, 643–652.CrossRefGoogle Scholar
  21. Mesquita, D. P., Amaral, A. L., & Ferreira, E. C. (2011b). Characterization of activated sludge abnormalities by image analysis and chemometric techniques. Analytica Chimica Acta, 705, 235–242.CrossRefGoogle Scholar
  22. Mesquita, D. P., Amaral, A. L., & Ferreira, E. C. (2013). Activated sludge characterization through microscopy: a review on quantitative image analysis and chemometric techniques. Analytica Chimica Acta, 802, 14–28.CrossRefGoogle Scholar
  23. Nicolau, A., Dias, N., Mota, M., & Lima, N. (2001). Trends in the use of protozoa in the assessment of wastewater treatment. Microbiology Research, 152, 621–630.CrossRefGoogle Scholar
  24. Nicolau, A., Neto, M. M., Santos, L., Fernandes, V., & Mota, M. (2015). PROTOFILWW: two year-sampling of protozoa, little metazoan and filamentous bacteria in 37 Portuguese wastewater treatment plants. Journal of Biotechnology, 208(Suppl), S18.CrossRefGoogle Scholar
  25. Pajdak-Stós, A., Sobczyk, M., Fiałkowska, E., Kocerba-Soroka, W., & Fyda, J. (2017). The effect of three different predatory ciliate species on activated sludge microfauna. European Journal of Protistology, 58, 87–93.CrossRefGoogle Scholar
  26. Parada-Albarracín, J. A., Pérez, J., & Gómez, M. A. (2017). Bioindicator value of flagellates in urban wastewater treatment using membrane bioreactors. Water Research, 122, 526–535.CrossRefGoogle Scholar
  27. Peng, Y., Li, J., Lu, J., Xiao, L., & Yang, L. (2018). Characteristics of microbial community involved in early biofilms formation under the influence of wastewater treatment plant effluent. Journal of Environmental Sciences, 66, 113–124.CrossRefGoogle Scholar
  28. Pérez-Uz, B., Arregui, L., Calvo, P., Salvadó, H., Fernández, N., Rodríguez, E., Zornoza, A., & Serrano, S. (2010). Assessment of plausible bioindicators for plant performance in advanced wastewater treatment systems. Water Research, 44, 5059–5069.CrossRefGoogle Scholar
  29. Poole, J. E. P. (1994). A study of the relationship between the mixed liquor fauna and plant performance for a variety of activated sludge sewage treatment works. Water Research, 18, 281–287.CrossRefGoogle Scholar
  30. Rice, E. W., Baird, R. B., Eaton, A. D. & Clesceri, L. S. (2012). Standard methods for the examination of water and wastewater. American Public Health Association, American Water Works Association, Water Environment Federation.Google Scholar
  31. Richard, M. (1991). Activated sludge microbiology. Virginia: The Water Pollution Control Federation.Google Scholar
  32. Salvadó, H., Gracia, M. P., & Amigó, J. M. (1995). Capability of ciliated protozoa as indicators of effluent quality in activated sludge plants. Water Research, 29, 1041–1050.CrossRefGoogle Scholar
  33. Santos, L. A., Ferreira, V., Neto, M. M., Pereira, M. A., Mota, M., & Nicolau, A. (2015a). Study of 16 Portuguese activated sludge systems based on filamentous bacteria populations and their relationships with environmental parameters. Applied Microbiology and Biotechnology, 99(12), 5307–5316.CrossRefGoogle Scholar
  34. Santos, L. A., Ferreira, V., Pereira, M. O., & Nicolau, A. (2015b). Relationship between protozoan and metazoan communities and operation and performance parameters in a textile sewage activated sludge system. European Journal of Protistology, 50(4), 319–328.CrossRefGoogle Scholar
  35. Singh, K. P., Malik, A., Mohan, D., Sinha, S., & Singh, V. K. (2005). Chemometric data analysis of pollutants in wastewater—a case study. Analytica Chimica Acta, 532, 15–25.CrossRefGoogle Scholar
  36. Tchobanoglous, G., Burton, F. L., & Stensel, H. D. (2003). Metcalf & Eddy wastewater engineering treatment disposal and reuse. New York: McGraw Hill.Google Scholar
  37. Tharrault, Y., Mourot, G. & Ragot, J. (2009) WWTP diagnosis based on robust principal component analysis. Proceedings of the 7 th IFAC Symposium on Fault Detection, Supervision and Safety of Technical Processes, 1342-1347.CrossRefGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2018

Authors and Affiliations

  1. 1.Instituto Politécnico de Coimbra, ISECCoimbraPortugal
  2. 2.CEB—Centre of Biological EngineeringUniversidade do MinhoBragaPortugal
  3. 3.CERNAS—Center of Studies on Natural Resources, Environment and SocietyInstituto Politécnico de Coimbra, ESACCoimbraPortugal
  4. 4.GERST/CIEPQPF—Faculty of Sciences and TechnologyUniversidade de Coimbra—Pólo IICoimbraPortugal

Personalised recommendations