Pierre Servais

Detection and enumeration of coliforms in drinking water: current methods and emerging approaches

The coliform group has been used extensively as an indicator of water quality and has historically led to the public health protection concept. The aim of this review is to examine methods currently in use or which can be proposed for the monitoring of coliforms in drinking water. Actually, the need for more rapid, sensitive and specific tests is essential in the water industry. Routine and widely accepted techniques are discussed, as are methods which have emerged from recent research developments.Approved traditional methods for coliform detection include the multiple-tube fermentation (MTF) technique and the membrane filter (MF) technique using different specific media and incubation conditions. These methods have limitations, however, such as duration of incubation, antagonistic organism interference, lack of specificity and poor detection of slow-growing or viable but non-culturable (VBNC) microorganisms. Nowadays, the simple and inexpensive membrane filter technique is the most widely used method for routine enumeration of coliforms in drinking water.The detection of coliforms based on specific enzymatic activity has improved the sensitivity of these methods. The enzymes beta-D galactosidase and beta-D glucuronidase are widely used for the detection and enumeration of total coliforms and Escherichia coli, respectively. Many chromogenic and fluorogenic substrates exist for the specific detection of these enzymatic activities, and various commercial tests based on these substrates are available. Numerous comparisons have shown these tests may be a suitable alternative to the classical techniques. They are, however, more expensive, and the incubation time, even though reduced, remains too long for same-day results. More sophisticated analytical tools such as solid phase cytometry can be employed to decrease the time needed for the detection of bacterial enzymatic activities, with a low detection threshold. Detection of coliforms by molecular methods is also proposed, as these methods allow for very specific and rapid detection without the need for a cultivation step. Three molecular-based methods are evaluated here: the immunological, polymerase chain reaction (PCR) and in-situ hybridization (ISH) techniques. In the immunological approach, various antibodies against coliform bacteria have been produced, but the application of this technique often showed low antibody specificity. PCR can be used to detect coliform bacteria by means of signal amplification: DNA sequence coding for the lacZ gene (beta-galactosidase gene) and the uidA gene (beta-D glucuronidase gene) has been used to detect total coliforms and E. coli, respectively. However, quantification with PCR is still lacking in precision and necessitates extensive laboratory work. The FISH technique involves the use of oligonucleotide probes to detect complementary sequences inside specific cells. Oligonucleotide probes designed specifically for regions of the 16S RNA molecules of Enterobacteriaceae can be used for microbiological quality control of drinking water samples. FISH should be an interesting viable alternative to the conventional culture methods for the detection of coliforms in drinking water, as it provides quantitative data in a fairly short period of time (6 to 8 h), but still requires research effort. This review shows that even though many innovative bacterial detection methods have been developed, few have the potential for becoming a standardized method for the detection of coliforms in drinking water samples.

Determination of the biodegradable fraction of dissolved organic matter in waters

Abstract Two bioassay procedures are proposed for determining biodegradable dissolved organic carbon (BDOC) in waters. Both involves sterile filtration of the sample, reinoculation with a natural assemblage of bacteria from the same origin as the sample, and incubation for at least 10 days in the dark at 20°C. In the first procedure, dissolved organic carbon (DOC) is followed, until a plateau is reached and the difference between initial and final DOC is taken as a measure of BDOC. In the second procedure, bacterial biomass and mortality rate are followed and the integrated flux of mortality during the incubation period is calculated and divided by the growth yield to give an estimate of BDOC. Both procedures provide closely concordant results. An example of application to the study of ozonation and biological activated carbon filtration in drinking water treatment is presented.

Does the High Nucleic Acid Content of Individual Bacterial Cells Allow Us To Discriminate between Active Cells and Inactive Cells in Aquatic Systems

ABSTRACT The nucleic acid contents of individual bacterial cells as determined with three different nucleic acid-specific fluorescent dyes (SYBR I, SYBR II, and SYTO 13) and flow cytometry were compared for different seawater samples. Similar fluorescence patterns were observed, and bacteria with high apparent nucleic acid contents (HNA) could be discriminated from bacteria with low nucleic acid contents (LNA). The best discrimination between HNA and LNA cells was found when cells were stained with SYBR II. Bacteria in different water samples collected from seven freshwater, brackish water, and seawater ecosystems were prelabeled with tritiated leucine and then stained with SYBR II. After labeling and staining, HNA, LNA, and total cells were sorted by flow cytometry, and the specific activity of each cellular category was determined from leucine incorporation rates. The HNA cells were responsible for most of the total bacterial production, and the specific activities of cells in the HNA population varied between samples by a factor of seven. We suggest that nucleic acid content alone can be a better indicator of the fraction of growing cells than total counts and that this approach should be combined with other fluorescent physiological probes to improve detection of the most active cells in aquatic systems.

Dynamics of bacterioplankton in oligotrophic and eutrophic aquatic environments: bottom-up or top-down control?

Measurements of bacterial biomass, production and mortality have been carried out in a large range of aquatic environments, including eutrophic and oligotrophic ones. The general trends of variations of bacterial biomass, size, specific growth rate and mortality rate in all these environments are examined. The overall flux of bacterial production is taken as an index of the flux of organic matter available to bacteria, thus characterizing the richness of the environment. Bacterial biomass is roughly proportional to richness, while mean cell size increases with it. The turnover rate of biomass, as revealed either by growth or by mortality rates, appears to be fairly independent of richness.These observations are compatible with a simple resource-limited (bottom-up controlled) model of the dynamics of bacterioplankton. On the other hand, they are in contradiction with the predictions of a predator-controlled (top-down controlled) model.

Impacts of pipe materials on densities of fixed bacterial biomass in a drinking water distribution system

Densities of fixed bacterial biomass were measured on different pipe materials (PVC, PE, cemented steel, asbestos-cement, cemented cast iron, tarred steel and grey iron) incubated in drinking waters from different sources (ground waters and a surface water) which had different characteristics (temperature, concentration of residual oxidant and content of biodegradable organic matter). Results showed that the densities of bacterial biomass fixed on plastic-based materials (PE and PVC) were the lowest. Densities of bacterial biomass fixed on gray iron were from 10 to 45 times higher than those measured on plastic-based materials. Cement-based materials had intermediate values. Pairs of cement and PVC coupons were incubated at 15 different locations in a distribution system, and there was a strong correlation between the amount of fixed bacterial biomass on these coupons, with the cement containing 2.6 times more biomass.

A nitrogen budget of the Scheldt hydrographical basin

Abstract A nitrogen budget including nitrite, nitrate, ammonium and organic nitrogen is presented for the western Scheldt estuary. The nitrogen entering the estuarine zone is evaluated from measurements of NO 2 − , NO 3 − , NH 4 + and organic nitrogen concentration at Rupelmonde. These results are part of 10 years survey (1973–1983) of water quality in the Scheldt estuary. The origin of this load in the Scheldt estuary is further investigated by the evaluation of the contribution of domestic, industrial sewages, agriculture and breeding in the nitrogenous load of the upper Scheldt drainage basin. Domestic load is evaluated from the watershed population. Industrial sewages are quantified by use of the evaluation of specific nitrogen spoilage by the various industries as a function of their number of workers. Nitrogen leaching of agricultural soils has been measured by determining the nitrogen concentration in small river draining agricultural areas, upstream any domestic or industrial discharges. Cattle-farming wastes are for the biggest part spread on soils. A fraction however is directly rejected in rivers. Denitrification in the tributaries of the Scheldt is important in the control of nitrate entering the estuarine zone. Its evaluation will be presented. In the estuarine part of the Scheldt (Rupelmonde-Vlissingen), the nitrogenous load is important due to the upstream load and to the sewages of the Antwerp district. These sewages (domestic, industrial, agricultural) have been evaluated as described above for the upper Scheldt basin. The important load carried at that moment by the Scheldt gives rise to an important bacterial activity which results in anaerobic conditions. Denitrification then takes place. This process reduces NO 3 − to N 2 O and N 2 , i.e. eliminates a substantial fraction of the nitrate load in the Scheldt. The importance of this process will be quantified both by measurement of in situ denitrifying activities and by analysis of NO 2 − + NO 3 − profiles in the river. When reoxidation of the water occurs by reaeration and mixing with well aerated seawater, the total mineral nitrogen has a conservative behaviour as indicated by the (straight) linear relationships between Σ N min and chlorinity, in spite of the primary production, bacterial activities and sediment influence. This conservative behaviour of Σ N min is used in this work for evaluating N min exportation by the Scheldt to the North Sea. The straight line relation extrapolated at low salinity gives a “fictive nitrogen concentration” in fresh water. The product of this “fictive concentration” and the upstream discharge gives an accurate evaluation of the exportation flux of mineral nitrogen to the sea. This work shows the predominant role of denitrification in tributaries of the drainage basin and in the estuary itself as a nitrogen sink which reduces the amount of nitrogen exported by the Scheldt to the North Sea. It is suggested that the pursuit of the present waste water treatment policy, only based on the elimination of the organic load without any tertiary treatment, could result in increasing the nitrogen output into the Belgian-Dutch coastal zones by a factor 2–3.

Impact of an intense combined sewer overflow event on the microbiological water quality of the Seine River.

For a better understanding of the short and mid-term impacts of a combined sewer overflow (CSO) on the microbiological quality of the receiving river, we studied the composition of a CSO discharge and monitored during several hours the changes in the concentration of fecal indicator bacteria (FIB) in the impacted river water mass. The CSO occurred at the Clichy outfall (Paris agglomeration, France) in summer 2008 as a result of the most intense rainfall of the year. In 6h, 578, 705 m(3) of sewage and 124 t of suspended matter (SM) were discharged into the Seine River. The CSO contained 1.5 × 10(6)E. coli and 4.0 × 10(5) intestinal enterococci per 100 mL on average, and 77% of the E. coli were attached to SM. It was estimated that 89% of the CSO discharge was contributed by surface water runoff, and that resuspension of sewer sediment contributed to ∼75% of the SM, 10-70% of the E. coli and 40-80% of the intestinal enterococci. Directly downstream from the CSO outfall, FIB concentrations in the impacted water mass of the Seine River (2.9 × 10(5)E. coli and 7.6 × 10(4) intestinal enterococci per 100 mL) exceeded by two orders of magnitude the usual dry weather concentrations. After 13-14 h of transit, these concentrations had decreased by 66% for E. coli and 79% for intestinal enterococci. This decline was well accounted for by our estimations of dilution, decay resulting from mortality or loss of culturability and sedimentation of the attached fraction of FIB.

Fecal coliform removal in wastewater treatment plants studied by plate counts and enzymatic methods.

Twelve wastewater treatment plants (WWTPs) were sampled in France and Belgium in 1999 and 2000 in order to estimate the fecal coliform (FC) removal efficiency of various types of treatment. Only one of these WWTPs was equipped with a specific step to eliminate microorganisms (UV disinfection preceded by sand filtration). FC abundance was measured in raw and treated sewage by plate counts on selective medium and rapid beta-D-glucuronidase (GLUase)-based assays. Removal of culturable FC was the most efficient in treatments with high retention time (activated sludge process with nitrification and denitrification, lagooning), in biofiltration and in the treatment with a tertiary disinfection step. GLUase activity measurements showed the same removal pattern as plate counts except for UV disinfection, where no reduction of GLUase activity was measured. Specific loads of culturable FC and GLUase activity, i.e. daily amounts of culturable FC or GLUase activity in sewage per inhabitant-equivalent, were calculated in raw and treated wastewater for the different WWTPs.

Mortality rates of autochthonous and fecal bacteria in natural aquatic ecosystems.

Bacterial mortality has been investigated in freshwater (River Seine) and in marine (North Sea) systems using a method based on following the disappearance of radioactivity from the DNA of assemblages of bacteria previously labeled with tritiated thymidine. Measurement of bacterial mortality of autochthonous and various types of fecal bacteria allowed direct comparisons between their respective first-order mortality rates. Mortality rates obtained for the different types of bacteria in the River Seine were, respectively, 7.9-33.9 x 10(-3)h(-1) for Escherichia coli, 12.2-29.2 x 10(-3)h(-1) for S. faecium and 7.0-18.3 x 10(-3)h(-1) for the autochthonous bacteria. In the Belgian coastal waters, these rates were 4.6-27.3 x 10(-3)h(-1) for E. coli, 6.0-22.0 x 10(-3)h(-1) for S. typhimurium, 10.0-18.9 x 10(-3)h(-1) for S. faecium and 1.0-13.9 x 10(-3)h(-1) for autochthonous bacteria. In both environments, the overall mortality rates of autochthonous and the different fecal bacteria were in the same order of magnitude and overall mortality rates of E. coli were on average about twice as high for autochthonous bacteria. Grazing by protozooplankton was the dominant process of mortality for fecal and autochthonous bacteria in both environments. Except in a few situations, grazing by protozooplankton was responsible for more than 90% of the overall mortality rate of fecal and autochthonous bacteria in the river and in the coastal area.

Impact of temperature on nitrification in biological activated carbon (BAC) filters used for drinking water treatment

The impact of temperature on nitrification in biological granular activated carbon (GAC) filters was evaluated in order to improve the understanding of the nitrification process in drinking water treatment. The study was conducted in a northern climate where very cold water temperatures (below 2 degrees C) prevail for extended periods and rapid shifts of temperature are frequent in the spring and fall. Ammonia removals were monitored and the fixed nitrifying biomass was measured using a method of potential nitrifying activity. The impact of temperature was evaluated on two different filter media: an opened superstructure wood-based activated carbon and a closed superstructure activated carbon-based on bituminous coal. The study was conducted at two levels: pilot scale (first-stage filters) and full-scale (second-stage filters) and the results indicate a strong temperature impact on nitrification activity. Ammonia removal capacities ranged from 40 to 90% in pilot filters, at temperatures above 10 degrees C, while more than 90% ammonia was removed in the full-scale filters for the same temperature range. At moderate temperatures (4-10 degrees C), the first stage pilot filters removed 10-40% of incoming ammonia for both media (opened and closed superstructure). In the full-scale filters, a difference between the two media in nitrification performances was observed at moderate temperatures: the ammonia removal rate in the opened superstructure support (more than 90%) was higher than in the closed superstructure support (45%). At low temperatures (below 4 degrees C) both media performed poorly. Ammonia removal capacities were below 30% in both pilot- and full-scale filters.