Bacteria permeabilisation and disruption caused by sludge reduction technologies evaluated by flow cytometry

Technologies proposed in the last decades for the reduction of the sludge production in wastewater treatment plants and based on the mechanism of cell lysis-cryptic growth (physical, mechanical, thermal, chemical, oxidative treatments) have been widely investigated at lab-, pilot- and, in some cases, at full-scale but the effects on cellular lysis have not always been demonstrated in depth. The research presented in this paper aims to investigate how these sludge reduction technologies affect the integrity and permeabilisation of bacterial cells in sludge using flow cytometry (FCM), which permits the rapid and statistically accurate quantification of intact, permeabilised or disrupted bacteria in the sludge using a double fluorescent DNA-staining instead of using conventional methods like plate counts and microscope.Physical/mechanical treatments (ultrasonication and high pressure homogenisation) caused moderate effects on cell integrity and caused significant cell disruption only at high specific energy levels. Conversely, thermal treatment caused significant damage of bacterial membranes even at moderate temperatures (45-55 °C). Ozonation significantly affected cell integrity, even at low ozone dosages, below 10 mgO3/gTSS, causing an increase of permeabilised and disrupted cells. At higher ozone dosages the compounds solubilised after cell lysis act as scavengers in the competition between soluble compounds and (particulate) bacterial cells. An original aspect of this paper, not yet reported in the literature, is the comparison of the effects of these sludge reduction technologies on bacterial cell integrity and permeabilisation by converting pressure, temperature and ozone dosage to an equivalent value of specific energy. Among these technologies, comparison of the applied specific energy demonstrates that achieving the complete disruption of bacterial cells is not always economically advantageous because excessive energy levels may be required. © 2010 Elsevier Ltd

Toxicant inhibition in activated sludge: Fractionation of the physiological status of bacteria

In wastewater treatment plants the sensitivity of activated sludge to a toxicant depends on the toxicity test chosen, and thus the use of more than one test is suggested. The physiological status of bacteria in response to toxicants was analysed by flow cytometry to distinguish intact, permeabilised, active cells and cells disrupted. Results were compared with respirometry and bioluminescence bioassay (Vibrio fischeri).3,5-Dichlorophenol (DCP) was used as reference xenobiotic. DCP has a strong effect on cellular integrity, causing an increase in permeabilised and disrupted cells. A reduction of 44-80% of intact cells with 6-30. mgDCP/L for 5. h was found. Inhibition of active cells was 25-49%, at 6-30. mgDCP/L for 5. h. The bioluminescence bioassay resulted oversensitive to DCP compared to tests based on activated sludge, while oxygen uptake rate was affected similarly to intact cells measured by flow cytometry. Landfill leachate was tested: a detrimental impact on both cellular integrity and enzymatic activity was observed. Reduction of intact cells and active cells was by 32% and 61% respectively after addition of 50% (v/v) of leachate for 5. h. The flow cytometry analysis proposed here might be widely applicable in the monitoring of various toxicants and in other aquatic biosystems

Bacteria viability and decay in water and soil of vertical subsurface flow constructed wetlands

In this study the functional status of bacterial biomass within a vertical subsurface flow (VSSF) constructed wetland was examined with the aim to understand the relationship between viable and dead bacteria in soil and influent/effluent wastewater and elucidate the large amount of dead cells in the soil which may affect the long-term behavior of the system. The quantification of viable and dead bacteria in influent and effluent wastewater and in the soil of a VSSF was performed at single-cell level by flow cytometry (FCM). An optimised pre-treatment was applied to soil samples using sodium pyrophosphate and ultrasonication at a specific energy of 80kJ/L. Viable and dead cells were detected on the basis of cellular membrane integrity coupling SYBR-Green I and Propidium Iodide. The bacteria profile in the VSSF soil depends on the depth and the material grain size. In the upper 0-10cm sand layer the number of total bacteria per gram of dry weight (DW) was higher (1.82×109 cells/gDW) than in the deeper 40-50cm (4.8×108 cells/gDW) probably due to the vertical feeding and a sieving effect of influent in the top layers. Bacterial biomass in the entire VSSF depth was 0.082mgVSS/gDW or 144gVSS/m3 (per cubic meter of VSSF bed). Size of viable bacteria in the VSSF was smaller (0.16μm3/cell) than typical size of activated sludge (0.23μm3/cell), due to lower nutrient conditions and a longer retention time of viable bacteria in the bed, estimated at around 130 days by mass balance. Dead bacteria were prevalent in the VSSF soil with a viable/dead bacteria ratio (V/D) of 0.52. The content of dead bacteria might be higher in the soil due to the presence of unsaturated zones not reached by fresh influent wastewater ("dead-zones"), where moisture and substrate are not so available and bacteria may die. Conversely, the higher V/D ratio (3.3) in the effluent reflects the enrichment of wastewater with viable bacteria during the passage through the VSSF bed and along preferential water flow, with higher water content and substrate availability, where the bacterial growth is favored

Direct quantification of bacterial biomass in influent, effluent and activated sludge of wastewater treatment plants by using flow cytometry

A rapid multi-step procedure, potentially amenable to automation, was proposed for quantifying viable and active bacterial cells, estimating their biovolume using flow cytometry (FCM) and to calculate their biomass within the main stages of a wastewater treatment plant: raw wastewater, settled wastewater, activated sludge and effluent. Fluorescent staining of bacteria using SYBR-Green I + Propidium Iodide (to discriminate cell integrity or permeabilisation) and BCECF-AM (to identify enzymatic activity) was applied to count bacterial cells by FCM. A recently developed specific procedure was applied to convert Forward Angle Light Scatter measured by FCM into the corresponding bacterial biovolume. This conversion permits the calculation of the viable and active bacterial biomass in wastewater, activated sludge and effluent, expressed as Volatile Suspended Solids (VSS) or particulate Chemical Oxygen Demand (COD). Viable bacterial biomass represented only a small part of particulate COD in raw wastewater (4.8 ± 2.4%), settled wastewater (10.7 ± 3.1%), activated sludge (11.1 ± 2.1%) and effluent (3.2 ± 2.2%). Active bacterial biomass counted for a percentage of 30-47% of the viable bacterial biomass within the stages of the wastewater treatment plant. © 2010 Elsevier Ltd

Surrogate parameters for the rapid microbial monitoring in a civil protection module used for drinking water production

Rapid analyses based on flow cytometry (FCM) and quantitative PCR (qPCR) were proposed and applied in a full-scale mobile water treatment plant (flow rate of 4.4. L/s) utilized as a civil protection module for drinking water production for quasi real-time monitoring. The rapid methods applied here are two cultivation-independent techniques (FCM and qPCR). The microbiological quality of water was monitored on the basis of alternative microbial parameters, detecting cells with an intact and permeabilised membrane (in 20. min), cells with β-. d-galactosidase activity (in 40. min) and Escherichia coli (E. coli, in less than 3. h). These rapid techniques were compared with some conventional culturable bacteria groups (aerobic mesophilic bacteria, total coliforms and E. coli).Although intact bacteria were two orders of magnitude higher than culturable aerobic mesophilic bacteria (due to a large fraction of viable-but-not-culturable cells, but also chemolithotrophic bacteria), they both showed not significant reduction in cells after filtration, 2-3. log of removal after ozonation and a regrowth of about 1. log after granular activated carbon. Cells with β-. d-galactosidase activity (belonging to the group of total coliforms) were higher than culturable total coliforms, due to a large presence of active-but-not-culturable cells, especially in ozone treated water.E. coli quantified by qPCR decreased through filtration and they were under the quantification limit after ozonation, analogously to culturable E. coli. Despite a higher quantification limit for FCM and qPCR, they appear sufficiently accurate and suitable as surrogate microbial parameters, considering their rapidity (about an half hour with FCM). In the case of strong stress conditions such as ozonation, the surrogate microbial parameters, which include viable-but-not-culturable cells, might result more sensible in the evaluation of treatment efficiency

Toxicant inhibition in activated sludge: Fractionation of the physiological status of bacteria

In wastewater treatment plants the sensitivity of activated sludge to a toxicant depends on the toxicity test chosen, and thus the use of more than one test is suggested. The physiological status of bacteria in response to toxicants was analysed by flow cytometry to distinguish intact, permeabilised, active cells and cells disrupted. Results were compared with respirometry and bioluminescence bioassay (Vibrio fischeri).3,5-Dichlorophenol (DCP) was used as reference xenobiotic. DCP has a strong effect on cellular integrity, causing an increase in permeabilised and disrupted cells. A reduction of 44-80% of intact cells with 6-30. mgDCP/L for 5. h was found. Inhibition of active cells was 25-49%, at 6-30. mgDCP/L for 5. h. The bioluminescence bioassay resulted oversensitive to DCP compared to tests based on activated sludge, while oxygen uptake rate was affected similarly to intact cells measured by flow cytometry. Landfill leachate was tested: a detrimental impact on both cellular integrity and enzymatic activity was observed. Reduction of intact cells and active cells was by 32% and 61% respectively after addition of 50% (v/v) of leachate for 5. h. The flow cytometry analysis proposed here might be widely applicable in the monitoring of various toxicants and in other aquatic biosystems