Long-amplicon propidium monoazide-PCR enumeration assay to detect viable Campylobacter and Salmonella.

AIMS: The effect of amplicon length on the ability of propidium monoazide-PCR (PMA-PCR) to reliably quantify viable cells without interference from dead cells was tested on heat- and ultraviolet (UV)-killed Salmonella enterica and Campylobacter jejuni, two important enteric pathogens of concern in environmental, food and clinical samples. METHODS AND RESULTS: PMA treatment followed by quantitative PCR (qPCR) amplification of short DNA fragments (<200 bp) resulted in incomplete signal inhibition of heat-treated Salm. enterica (3 log reduction) and Camp. jejuni (1 log reduction), whereas PCR amplification of a long DNA fragment (1·5 and 1·6 kb) completely suppressed the dead cell signal. PMA pretreatment of UV-irradiated cells did not affect PCR amplification, but long-amplicon PCR was shown to detect only viable cells for these samples, even without the addition of PMA. CONCLUSIONS: The long-amplicon PMA-PCR method was effective in targeting viable cells following heat and UV treatment and was applicable to enteric pathogens including Salmonella and Campylobacter that are difficult to enumerate using culture-based procedures. SIGNIFICANCE AND IMPACT OF THE STUDY: PCR amplicon length is important for effective removal of the dead cell signal in PMA pretreatment methods that target membrane-damaged cells, and also for inactivation mechanisms that cause direct DNA damage.

The occurrence of Campylobacter in river water and waterfowl within a watershed in southern Ontario, Canada.

AIMS: Quantitative PCR and a culture method were used to investigate Campylobacter occurrence over 3 years in a watershed located in southern Ontario, Canada that is used as a source of drinking water. METHODS AND RESULTS: Direct DNA extraction from river water followed by quantitative PCR analysis detected thermophilic campylobacters at low concentrations (<130 cells 100 ml(-1) ) in 57-79% of samples taken from five locations. By comparison, a culture-based method detected Campylobacter in 0-23% of samples. Water quality parameters such as total Escherichia coli were not highly correlated with Campylobacter levels, although higher pathogen concentrations were observed at colder water temperatures (<10°C). Strains isolated from river water were primarily nalidixic acid-susceptible Campylobacter lari, and selected isolates were identified as Campylobacter lari ssp. concheus. Campylobacter from wild birds (seagulls, ducks and geese) were detected at a similar rate using PCR (32%) and culture-based (29%) methods, and although Campylobacter jejuni was isolated most frequently, C. lari ssp. concheus was also detected. CONCLUSIONS: Campylobacter were frequently detected at low concentrations in the watershed. Higher prevalence rates using quantitative PCR was likely because of the formation of viable but nonculturable cells and low recovery of the culture method. In addition to animal and human waste, waterfowl can be an important contributor of Campylobacter in the environment. SIGNIFICANCE AND IMPACT OF THE STUDY: Results of this study show that Campylobacter in surface water can be an important vector for human disease transmission and that method selection is important in determining pathogen occurrence in a water environment.

Decision support systems in water and wastewater treatment process selection and design: a review.

The continuously changing drivers of the water treatment industry, embodied by rigorous environmental and health regulations and the challenge of emerging contaminants, necessitates the development of decision support systems for the selection of appropriate treatment trains. This paper explores a systematic approach to developing decision support systems, which includes the analysis of the treatment problem(s), knowledge acquisition and representation, and the identification and evaluation of criteria controlling the selection of optimal treatment systems. The objective of this article is to review approaches and methods used in decision support systems developed to aid in the selection, sequencing of unit processes and design of drinking water, domestic wastewater, and industrial wastewater treatment systems. Not surprisingly, technical considerations were found to dominate the logic of the developed systems. Most of the existing decision-support tools employ heuristic knowledge. It has been determined that there is a need to develop integrated decision support systems that are generic, usable and consider a system analysis approach.

A comparison of DNA repair and survival of Escherichia coli O157:H7 following exposure to both low- and medium-pressure UV irradiation.

DNA repair and survival of pathogenic E. coli O157:H7 was investigated following exposure to ultraviolet (UV) radiation from both low-pressure (LP) and medium-pressure (MP) lamps. This study included irradiation at UV doses used in drinking water treatment and lower doses indicative of potential treatment problems. Immediately following UV exposure, an average log inactivation of 4.5 or greater was observed following all tested doses of LP (5, 8, 20 and 40 mJ/cm(2)) or MP UV (5 and 8 mJ/cm(2)) indicating the sensitivity of E. coli O157:H7 to UV irradiation. Following conditions conducive to repair, maximum photo repair occurred rapidly within 30 minutes after low doses (5 and 8 mJ/cm(2)) of LP UV. The rate of repair was much higher than reported previously in non-pathogenic E. coli (which occurred within 2 hours). In contrast to LP UV, limited photo repair of E. coli O157:H7 was observed following MP UV exposure at reduced doses (5 and 8 mJ/cm(2)). At these lower doses, low levels of light independent repair were observed following LP UV, but not following exposure of MP UV irradiation. This study indicates that MP UV may enhance UV disinfection of E. coli O157:H7 by reducing the ability to repair following non-ideal treatment conditions. Following doses used in drinking water treatment (20 and 40 mJ/cm(2)), low levels of photo repair following LP UV were evident.

Impact of support media in an integrated biofilter-submerged membrane system.

The impact of a support material on an integrated biofilter-membrane system, simulating a difficult-to-treat surface water, was examined in terms of membrane fouling rate and water quality parameters. The support media in the membrane tanks did not generally affect any of the water quality parameters measured; however, there was an observable difference in the membrane fouling rates between the two processes with the support media system fouling at least two times slower than the non-support system. Total organic carbon (TOC) removals at around 60% were observed for two integrated biofilter-immersed membrane processes with the majority of the TOC removal occurring in the biofilters. One of the membrane tanks contained a support media (Process A) while the other did not (Process B). The feedwater contained humic acid (65% w/w) and readily biodegradable carbons (35% w/w) in the forms of acetic acid, formic acid and formaldehyde. The influent TOC values were between 3.35 and 3.94 mg/L. Acetate removals varied between 66 and 83%, while over 90% of the formate was removed and the formaldehyde was completely removed in the biofilters. There was a decrease in the UV absorbance values by over 70% for both processes.

Effects of temperature and biodegradable organic matter on control of biofilms by free chlorine in a model drinking water distribution system.

This study used annular reactors (AR) to investigate, under controlled laboratory conditions, the effects of temperature and biodegradable organic matter (BOM) on the free chlorine residual needed to control biofilm accumulation, as measured by heterotrophic plate count (HPC) bacteria. Biofilm was grown on PVC coupons, initially in the absence of chlorine, at 6, 12, and 18 degrees C, in the presence and absence of a BOM supplement (250 microg C/L) added as acetate. During the early stages of chlorine addition, when no measurable free chlorine residual was present, a reduction in biofilm HPC numbers was observed. Subsequently, once sufficient chlorine was added to establish a residual, the biofilm HPC numbers expressed as log CFU/cm2 fell exponentially with the increase in free chlorine residual. Temperature appeared to have an important effect on both the chlorine demand of the system and the free chlorine residual required to control the biofilm HPC numbers to the detection limit (3.2 Log CFU/cm2). For the water supplemented with BOM, a strong linear correlation was found between the temperature and the free chlorine residual required to control the biofilm. At 6 degrees C, the presence of a BOM supplement appeared to substantially increase the level of free chlorine residual required to control the biofilm. The results of these laboratory experiments provide qualitative indications of effects that could be expected in full-scale systems, rather than to make quantitative predictions.

Removal of geosmin and 2-methylisoborneol by biological filtration.

The quality of drinking water is sometimes diminished by the presence of certain compounds that can impart particular tastes or odours. One of the most common and problematic types of taste and odour is the earthy/musty odour produced by geosmin (trans-1, 10-dimethyl-trans-9-decalol) and MIB (2-methylisoborneol). Taste and odour treatment processes including powdered activated carbon, and oxidation using chlorine, chloramines, potassium permanganate, and sometimes even ozone are largely ineffective for reducing these compounds to below their odour threshold concentration levels. Ozonation followed by biological filtration, however, has the potential to provide effective treatment. Ozone provides partial removal of geosmin and MIB but also creates other compounds more amenable to biodegradation and potentially undesirable biological instability. Subsequent biofiltration can remove residual geosmin and MIB in addition to removing these other biodegradable compounds. Bench scale experiments were conducted using two parallel filter columns containing fresh and exhausted granular activated carbon (GAC) media and sand. Source water consisted of dechlorinated tap water to which geosmin and MIB were added, as well as, a cocktail of easily biodegradable organic matter (i.e. typical ozonation by-products) in order to simulate water that had been subjected to ozonation prior to filtration. Using fresh GAC, total removals of geosmin ranged from 76 to 100% and total MIB removals ranged from 47% to 100%. The exhausted GAC initially removed less geosmin and MIB but removals increased over time. Overall the results of these experiments are encouraging for the use of biofiltration following ozonation as a means of geosmin and MIB removal. These results provide important information with respect to the role biofilters play during their startup phase in the reduction of these particular compounds. In addition, the results demonstrate the potential biofilters have in responding to transient geosmin and MIB episodes.

Integrated biofilter-immersed membrane system for the treatment of humic waters.

An integrated biofilter-immersed membrane study was conducted to determine the effect of placing a biofilter before or after a membrane for the treatment of a humic type water. The parameters measured included total organic carbon (TOC), organic acids, and specific ultraviolet absorbance (SUVA). The difference in membrane fouling and microbial growth for the two configurations was also examined. Greater TOC and organic acid removal occurred when the biofilter was located ahead of the membrane. The greatest decrease in SUVA values was associated with the membrane. The membrane located after the biofilter fouled at a slower rate than the membrane ahead of the biofilter. Fouling was slower when turbidity was present in the synthetic feedwater. A new method is proposed for determining the operational cleaning frequency of membranes, using an empirical model.

Inactivation and potential repair of Cryptosporidium parvum following low- and medium-pressure ultraviolet irradiation.

This study investigated the level of inactivation and the potential for Cryptosporidium parvum to repair following low doses (1 and 3mJ/cm(2)) of ultraviolet (UV) irradiation from both low- and medium-pressure UV lamps. Cryptosporidium parvum oocysts suspended in phosphate buffered saline were exposed to UV using a bench-scale collimated beam apparatus. Oocyst suspensions were incubated at 5 degrees C or 25 degrees C under light and dark conditions up to 120 h (5 days) following exposure to UV irradiation, to examine photoreactivation and dark repair potential, respectively. Cryptosporidium parvum infectivity was determined throughout the incubation period using an HCT-8 cell culture and an antibody staining procedure for detection. No detectable evidence of repair was observed after incubation under light or dark conditions following either LP or MP UV lamp irradiation.

Endotoxin inactivation in water by using medium-pressure UV lamps.

Deionized water was spiked with various concentrations of endotoxin and exposed to UV irradiation from medium-pressure UV lamps to assess endotoxin inactivation. It was found that endotoxin inactivation was proportional to the UV dose under the conditions examined. The inactivation rate was determined to be approximately 0.55 endotoxin unit/ml per mJ/cm(2) of irradiation delivered.

A fatal waterborne disease epidemic in Walkerton, Ontario: comparison with other waterborne outbreaks in the developed world.

An estimated 2,300 people became seriously ill and seven died from exposure to microbially contaminated drinking water in the town of Walkerton, Ontario, Canada in May 2000. The severity of this drinking water disaster resulted in the Government of Ontario calling a public inquiry by Mr. Justice Dennis O'Connor to address the cause of the outbreak, the role (if any) of government policies in contributing to this outbreak and, ultimately, the implications of this experience on the safety of drinking water across the Province of Ontario. The circumstances surrounding the Walkerton tragedy are an important reference source for those concerned with providing safe drinking water. Although some circumstances are obviously specific to this epidemic, others are uncomfortably reminiscent of waterborne outbreaks that have occurred elsewhere. These recurring themes suggested the need for attention to broad issues of drinking water security and they present the challenge for how drinking water safety can be managed to prevent such tragedies in the future.

Removal of easily biodegradable organic compounds by drinking water biofilms: analysis of kinetics and mass transfer.

This paper evaluates the rate of utilization of easily biodegradable organic compounds by drinking water biofilms. Tap water, which had been filtered through biologically active granular activated carbon, was used as an innoculum for biofilm growth in annular reactors (ARs). Synthetic cocktails of easily biodegradable material in the concentration range of 50-2,000 mgC/m3 were used as substrate for biofilm growth. Influent and effluent aggregate concentrations of biodegradable organic matter (BOM) were calculated by adding the measurable BOM components on a mass carbon basis. The aggregate BOM values were used for calculating the observed Damköhler number and Theile modulus (based on a reaction rate per unit surface area), which were used to determine whether external or internal mass transfer limited BOM removal. For all of the experimental trials, it was shown that neither external nor internal mass transfer limited BOM removal. Because the biofilms in this research are thin and the fact that mass transfer is not limiting, it was assumed that the bulk BOM concentration was approximately equal to the average BOM concentration in the biofilm. A linear model was obtained for the aggregate BOM flux and the product of the effluent BOM concentration and the biofilm density. The slope or the areal biodegradation rate (ka) for the aggregate BOM was 0.033 m/h, as determined through a linear regression.

Measurement of biomass activity in drinking water biofilters using a respirometric method.

A simple respirometric method was developed and applied for the measurement of biomass activity in bench-scale drinking water biofilters. The results obtained with the new method, i.e. biomass respiration potential (BRP), indicated a high sensitivity allowing the quantification of the activity of low amounts of biomass. The analysis of duplicate samples showed a reasonable reproducibility, i.e. average coefficient of variation of 14% (n = 19). The calculation of the ratio between biomass activity and the amount of viable biomass (phospholipid) at different filter depths indicated a substantial increase of this ratio with filter depth. This indicated an increased biomass activity per unit amount of viable biomass deeper in the biofilters, where biofilm thickness is low. The comparison of the filter profiles of biomass activity and dissolved biodegradable organic matter (BOM), expressed as theoretical oxygen demand, showed a high correlation between these profiles. Consequently, BRP results appear to be good indicators of the BOM removal capacity of the filter biomass. Therefore, BRP results can potentially be used in certain cases instead of BOM measurements for the assessment of the BOM removal capacity of drinking water biofilters, operated under different conditions. This is important because of the relative complexity of the measurements of BOM surrogates, e.g. assimilable organic carbon and biodegradable dissolved organic carbon, and BOM components.

Quantitative determination of oxalate and other organic acids in drinking water at low microgram/1 concentrations.

In this research, a recently developed ion chromatography method for organic acids was expanded to include oxalate. A major challenge was that oxalate elutes between inorganic anions such as sulfate, phosphate, bromide and nitrate, which are often present in much higher concentrations than oxalate. Optimization of the previously reported method made it possible to determine oxalate in these matrices. However, for those samples in which higher inorganic anion concentrations caused the oxalate peak to be obscured, a "heart-cut" column switching technique was used as an alternative. The method detection limit for oxalate was 9 micrograms/l with the direct approach and 6 micrograms/l for the "heat-cut" technique. These modifications represent a valuable supplement to a recently developed method for monitoring ozonation by-products in drinking water.

Determination of short-chain aliphatic, oxo- and hydroxy-acids in drinking water at low microgram per liter concentrations.

A fast and reliable ion chromatography method has been developed and applied to study the formation and consumption of organic acid ozonation by-products in a drinking water treatment plant. Water samples are injected directly into the ion chromatograph using a large sample loop (740 mu l) without any sample preparation step other than possibly filtration. Organic and inorganic anions are determined by separation on a high-capacity anion-exchange column followed by conductivity detection. The average recovery for the organic acids investigated (beta-hydroxybutyric, acetic, glycolic, butyric, formic, alpha-ketobutyric and pyruvic acid) ranged from 96 to 105%, and their method detection limits ranged from 1 to 5 mu g/l. When applied to samples taken from a drinking water treatment plant, the method proved to be reliable.

Biodegradation of aquatic organic matter with reference to drinking water treatment.

This paper presents results obtained in a pilot scale investigation of biological treatment for preparation of drinking water from the North Saskatchewan River at Edmonton, Canada. Although the concentration of natural organic matter (NOM) in the raw water varied substantially over the study period, parameters measuring the biodegradability and reactivity to chlorine of the NOM were closely correlated to the NOM concentration. As a result of treatment including ozonation and adsorption, two patterns of response emerged. Some parameters decreased through each step of the treatment process while others usually increased following ozonation and then decreased through subsequent steps. The levels of this latter group were decreased by biological activity in the filters (except for one treatment stream) and further reduced in the biologically active granular activated carbon (GAC) contactors.

Microbial release of 226Ra2+ from (Ba,Ra)SO4 sludges from uranium mine wastes.

226Ra2+ is removed from uranium mine effluents by coprecipitation with BaSO4. (Ba,Ra)SO4 sludge samples from two Canadian mine sites were found to contain active heterotrophic populations of aerobic, anaerobic, denitrifying, and sulfate-reducing bacteria. Under laboratory conditions, sulfate reduction occurred in batch cultures when carbon sources such as acetate, glucose, glycollate, lactate, or pyruvate were added to samples of (Ba,Ra)SO4 sludge. No external sources of nitrogen or phosphate were required for this activity. Further studies with lactate supplementation showed that once the soluble SO4(2-) in the overlying water was depleted, Ba2+ and 226Ra2+ were dissolved from the (Ba,Ra)SO4 sludge, with the concurrent production of S2-. Levels of dissolved 226Ra2+ reached approximately 400 Bq/liter after 10 weeks of incubation. Results suggest that the ultimate disposal of these sludges must maintain conditions to minimize the activity of the indigenous sulfate-reducing bacteria to ensure that unacceptably high levels of 226Ra2+ are not released to the environment.