Assessment of physiological state of microorganisms in activated sludge with flow cytometry: application for monitoring sludge production minimization

Abstract

Many sludge reduction processes have been studied for the minimization of sludge production in biological wastewater treatment. The investigations on most of these processes have monitored the increase of the soluble chemical oxygen demand, the sludge mass reduction, or the decrease of the floc size, but little information has been obtained on cell lysis and the change of the biological cell activity. However, employing any strategy for reducing sludge production may have an impact of microbial community in biological wastewater treatment process. This impact may influence the sludge characteristics and the quality of effluent. The objective of this study concerns the determination of the physiological state of activated sludge microorganisms during a sludge minimization process. A thermal treatment at 80 degrees C for 5, 20, 40 and 60 min was chosen in this study. Staining bacteria with CTC and SYTOX green was used to evaluate biological cell activity and viability of cell types contained in activated sludge, respectively. The monitoring of cell activity and viability was performed using flow cytometry (FCM) analysis before and after thermal treatment of activated sludge. Results indicated an increase in the number of permeabilized cells and a decrease in the number of active cells, subsequent to the thermal treatment. The study also confirms the potential of FCM to successfully evaluate the physiological heterogeneity of an activated sludge bacterial population. Moreover, the experimentally observed correlations between the FCM results and the organic matter solubilization in activated sludge samples during thermal treatment revealed that the increase in the soluble organic matter concentration was predominantly due to an intracellular material release. Identifying the increase in activated sludge hydrolysis requires a precise knowledge of the involved mechanisms, and this study indicated that the FCM, used in conjunction with specific probes, could be a useful tool.

Evaluation of the microbial cell structure damages in alkaline pretreatment of waste activated sludge

“…The damages of microbial cell walls were evaluated in terms of the N-acetylglucosamine content (one of cell wall hydrolysates) , which was detected via the Morgan-Elson colorimetric method with glucosamine as the standard (Morgan and Elson, 1934). The damages of microbial cell membranes were evaluated via flow cytometry (FCM; FACSCalibur, BD Pharmingen, Heidelberg, Germany) (Prorot et al, 2008;Guo et al, 2014). The damages of microbial cell nuclei were evaluated in terms of the DNA content, which was measured via the diphenylamine colorimetric method by using fish sperm DNA and sodium salt as the standard (Sun et al, 1999).…”

Flow Cytometry as a Powerful Tool for Monitoring Microbial Population Dynamics in Sludge

“…One promising tool of flow cytometry consists of characterising and distinguishing different the physiological states of microorganisms at the single-cell level (Joux & Lebaron, 2000) (Nebevon-Caron et al, 2000). The ability of flow cytometry to distinguish between different physiological states is important for assessing the growth of microorganisms in oligotrophic environments (Berney et al, 2007), the survival of pathogenic microorganisms (Vital et al, 2007) and the effects of bactericidal treatments or different environmental stresses on microbial activity (Prorot et al, 2008) (Ziglio et al, 2002) (Foladori et al, 2010a) (Booth, Ian R, 2002). When employed in conjunction with fluorescent dyes, flow cytometry is able to measure various biological parameters (i.e., nucleic acid content, metabolic activity, enzyme activity and membrane integrity), allowing the detection of microorganisms at viable, viable but non-cultivable (or intermediate) and non-viable states (Joux & Lebaron, 2000) (Walberg et al, 1999).…”

“…FCM gained interest with its application to industrial microbial processes (Díaz et al, 2010). Other fields of application have appeared to be of interest, such as the optimisation of SRP (sludge reduction processes) (Prorot et al, 2008;Prorot et al, 2011).…”

“…The disruption of microorganisms is not so easily determined by the analysis of particle size because disrupted cell walls and the original cells are of a similar size (Müller et al, 2004). For this reason, the use of FCM -which allows a rapid and accurate quantification of the total bacterial population -could provide very specific and useful information about the physiological state of bacteria, including cell disruption (Prorot et al, 2008)) during sludge hydrolysis treatment. Foladori et al (2007) investigated the effect of the sonication treatment on the viability of bacteria present at different points in a WWTP using FCM after fluorescent nucleic acid staining (SYBR-Green and propidium iodide).…”

“…They concluded that in activated sludge samples, low levels of specific ultrasonic energy (Es) produced a prevalent disaggregation of flocs releasing single cells in the bulk liquid, while the disruption of the bacteria was induced only by very high levels of Es (Es>120 kJ L-1). Prorot et al (2008) assessed the possibility of using FCM to evaluate the physiological state changes of bacteria occurring during sludge thermal treatment. To this end, they stained bacteria with CTC and SYTOX green was used to evaluate biological cell activity and the viability of cell types contained in the activated sludge.…”