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Assessment of total bacterial cells in extended aeration activated sludge plants using flow cytometry as a microbial monitoring tool

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Abstract

The extended aeration activated sludge (EAAS) process is one of the most applied biological processes in small towns. Here, we study the abundance and viability of total bacterial cells in two wastewater treatment plants (WWTPs) operating with an EAAS process. We use flow cytometry (FCM) combined with SYTO13 and propidium iodide (PI) dyes as a rapid, easy, reliable and accurate microbial monitoring tool. A disaggregation procedure with an ultrasonic bath was designed to detach total bacterial cells from activated sludge flocs for subsequent FCM analysis. This procedure permitted the recovery of total bacterial cells from sludge flocs without affecting bacterial viability, as indicated by bacterial strain controls. Since FCM is a multi-parameter technique, it was possible to determine total bacterial abundance and their viability in the activated sludge. As a comparative method, epifluorescence microscopy was also used to quantify total bacterial cells; both methods produced similar results. The FCM analysis revealed relative microbial stability in both the WWTPs. The total bacterial abundance quantified by FCM in the two plants studied was 1.02–6.23 × 1011 cells L−1 with 70–72 % viability, one logarithm less than that reported in the literature for WWTPs using the conventional activated sludge process. This can be explained by the difference in the operational parameters between the conventional plant and EAAS, mainly the organic loading rate.

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Abbreviations

FCM:

Flow cytometry

WWTP:

Wastewater treatment plant

EAAS:

Extended aeration activated sludge

F/M:

Food to microorganisms

BOD5 :

5-day biochemical oxygen demand

MLSS:

Mixed liquor suspended solids

EPS:

Extracellular polymeric substances

DAPI:

4′, 6-diamidino-2-phenylindole

PI:

Propidium iodide

V/V:

Volume/volume

EM:

Epifluorescence microscopy

ANOVA:

Analysis of variance

COD:

Chemical oxygen demand

HRT:

Hydraulic retention time

Max:

Maximum value

Min:

Minimum value

SD:

Standard deviation

X 2 :

Chi-square

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Acknowledgments

The authors thank the ACCIONA Agua Company for funding this study and especially Mr. Amador Rancaño. The authors also thank Dr. Jaume Comas Riu of the Centres Científics i Tecnològics de la Universitat de Barcelona (CCiTUB) for his help with FCM data analysis, Dr. Javier Méndez Viera for his collaboration, Mr. Pedro Aguiló and Victor Barreiro of AGBAR, Empresa Metropolitana de Gestió del Cicle Integral de l′Aigua, S.A.

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As authors, we declare that we have no conflict of interest.

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Correspondence to Rosa M Araujo.

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Responsible editor: Gerald Thouand

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Abzazou, T., Salvadó, H., Bruguera-Casamada, C. et al. Assessment of total bacterial cells in extended aeration activated sludge plants using flow cytometry as a microbial monitoring tool. Environ Sci Pollut Res 22, 11446–11455 (2015). https://doi.org/10.1007/s11356-015-4372-3

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