Abstract
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.
Highlights
Water filtration, with sand or granular activated carbon (GAC), is a conventional treatment process widely used in traditional drinking water treatment plants, whereby raw water is passed through a porous bed of filter medium in order to remove fine particles and soluble organic matter
The highest recovery was obtained with the solution of TWEENPP, where after one cycle of low energy sonication (LES) and two cycles of high energy sonication (HES) 24 ± 1% more cells were extracted compared to TAP (Fig. 1a)
Autoclaved TAP water seemed to release cells in two distinct phases: half of the cells were released in the first two steps (53% of cells released during addition and LES), 40% were released in the HES1
Summary
With sand or granular activated carbon (GAC), is a conventional treatment process widely used in traditional drinking water treatment plants, whereby raw water is passed through a porous bed of filter medium in order to remove fine particles and soluble organic matter. Studying microbial diversity in water filters is an exciting new research direction, but the accurate quantification of bacteria in these filters is important. It is an often under-appreciated, complementary element of microbial ecology, which is essential for determining bacterial growth rates and substrate utilisation kinetics, for theoretical modelling (Meynet et al, 2014, 2012), mass balances (Vignola et al, 2018) and for comprehensive interpretation of microbiome surveys (Props et al, 2017). The search for rapid and reliable techniques to estimate microbial cell numbers in diverse environments, and in filter media has become a scientific priority (Davis, 2014)
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