Flow-cytometric quantification of microbial cells on sand from water biofilters

Vignola, M. , Werner, D., Hammes, F., King, L. C. and Davenport, R. J. (2018) Flow-cytometric quantification of microbial cells on sand from water biofilters. Water Research, 143, pp. 66-76. (doi: 10.1016/j.watres.2018.05.053) (PMID:29940363)

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Rapid quantification of absolute microbial cell abundances is important for a comprehensive interpretation of microbiome surveys and crucial to support theoretical modelling and the design of engineered systems. In this paper, we propose a protocol specifically optimised for the quantification of microbial abundances in water biofilters using flow cytometry (FCM). We optimised cell detachment from sand biofilter particles for FCM quantification through the evaluation of five chemical dispersants (NaCl, Triton-X100, CaCl2, sodium pyrophosphate (PP), Tween 80 combined with PP), different mechanical pre-treatments (low and high energy sonication and shaking) and two fixation methods (glutaraldehyde and ethanol). The developed protocol was cross-compared using other established and commonly employed methods for biomass quantification in water filter samples (adenosine triphosphate (ATP) quantification, real-time quantitative PCR (qPCR) and volatile solids (VS)). The highest microbial count was obtained by detaching the biofilm from biofilter grains and dispersing clusters into singles cells using Tween 80 and sodium pyrophosphate combined with four steps of high energy sonication (27W, for 80 s each step); glutaraldehyde was shown to be the best fixative solution. The developed protocol was reliable and highly reproducible and produced results that are comparable to data from alternative quantification methods. Indeed, high correlations were found with trends obtained through ATP and qPCR (ρ = 0.98 and ρ = 0.91) measurements. The VS content was confirmed as an inaccurate method to express biomass in sand samples since it correlated poorly with all the other three methods (ρ = 0.005 with FCM, 0.002 with ATP and 0.177 with qPCR). FCM and ATP showed the strongest agreement between absolute counts with a slope of the correlation equal to 0.7, while qPCR seemed to overestimate cell counts by a factor of ten. The rapidity and reproducibility of the method developed make its application ideal for routine quantification of microbial cell abundances on sand from water biofilters and thus useful in revealing the ecological patterns and quantifying the metabolic kinetics involved in such systems.

Item Type:Articles
Additional Information:This study was supported by MERMAID, a Marie Skłodowska-Curie Initial Training Network [grant number 607492]. Russell Davenport would also like to acknowledge a Challenging Engineering award from the Engineering and Physical Sciences Research Council (EP/I025782/1).
Glasgow Author(s) Enlighten ID:Vignola, Dr Marta
Authors: Vignola, M., Werner, D., Hammes, F., King, L. C., and Davenport, R. J.
College/School:College of Science and Engineering > School of Engineering > Infrastructure and Environment
Journal Name:Water Research
ISSN (Online):1879-2448
Published Online:29 May 2018
Copyright Holders:Copyright © 2018 The Authors
First Published:First published in Water Research 143:66-76
Publisher Policy:Reproduced under a Creative Commons License

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