Hybridisation of F+ RNA coliphages detected in shellfish samples with oligonucleotide probes to assess the origin of microbiological pollution of shellfish.

Current measures for controlling the public health risks associated with bivalve molluscan shellfish consumption rely on the use of Escherichia coli to indicate the sanitary quality of shellfish harvesting areas. However, it has been demonstrated that E. coli is an inadequate indicator of the viral risk associated with shellfish. An alternative indicator, male-specific B+ coliphages, have been investigated as viral indicators of faecal contamination that may provide source-specific information for impacted environmental waters. This study compared the distribution of E. coli and F+ RNA bacteriophages in shellfish grown in harvesting areas of Greece and also examined the presence and proportions of the different subgroups of F+ RNA coliphages in shellfish. F+ RNA bacteriophages were present in shellfish at higher concentrations than E. coli. Elevated numbers of F+ RNA bacteriophages observed in the winter concur with the known increased viral risk associated with shellfish harvested at that time of year in Greece. The majority of F+ RNA coliphages detected in shellfish samples belonged to group IV which indicated the possible presence of animal faecal material in sample harvesting areas. Phages of groups II and III (human waste and human faecal material, respectively) were present at low levels. Finally, 8% of the phages hybridised were found to belong to group I. The presence of group IV showed seasonal distribution (more in winter, less in summer) whereas the other groups did not show any difference. Monitoring of F+ coliphage subgroups may indicate the presence and major sources of microbial inputs to surface waters; however, environmental effects on the relative occurrence of different groups need to be considered.     

Pathogen removal efficiency from UASB + BF effluent using conventional and UV post-treatment systems.

The aim of this study was to verify the efficiency of removal of microorganisms in effluents of a Wastewater Treatment Plant (WWTP) comprising an association of a UASB reactor followed by three submerged aerated biofilters (BAF) and one tertiary filter. The WWTP designed to treat domestic wastewater from a population of 1,000 inhabitants showed high removal efficiency for organic matter and suspended solids. Helminth eggs were also efficiently removed from the tertiary effluent and were found in the sludge from the UASB reactor; however, removal of bacteria in this system was very low. To enhance the efficiency of the system, the effluent from tertiary filters was submitted to UV disinfection in a real scale reactor. Our results showed that UV irradiation was very effective at lowering the concentrations of E. coli, thermotolerant coliforms and coliphages to acceptable levels for agricultural reuse. Salmonella spp. and helminth eggs were seeded into the tertiary effluent before passing through the UV reactor. Salmonella was not found in the final effluent, but helminth eggs were not completely inactivated by UV irradiation and viable eggs were detected after 28 d of incubation.     

Contribution of the colmation layer to the elimination of coliphages by slow sand filtration.

River bank or slow sand filtration is a major procedure for processing surface water to drinking water in central europe. In order to model the performance of river bank and slow sand filtration plants, we are studying the different mechanisms by which the elimination of pathogens is realized. An important question concerning the mode of action of slow sand filters and river bank filtration units is the role of the colmation layer or "schmutzdecke" on the elimination of human pathogens. The schmutzdecke is an organic layer which develops at the surface of the sand filter short after the onset of operation. We have inoculated a pilot plant for slow sand filtration with coliphages and determined their rate of breakthrough and their final elimination. In the first experiment, with a colmation layer still missing, the breakthrough of the coliphages in the 80 cm mighty sandy bed amounted to ca. 40 %. In contrast, less than 1 % of coliphages escaped from the filter as the same experiment was repeated two months later, when a substantial colmation layer had developed. Our preliminary conclusions are that the colmation layer is extremely efficient in eliminating of viruses.     

Characterisation of the biosand filter for E. coli reductions from household drinking water under controlled laboratory and field use conditions.

More than a billion people in the developing world lack access to safe and reliable sources of drinking water. Point of use (POU) household water treatment technology allows people to improve the quality of their water by treating it in the home. One emerging POU technology is the biosand filter (BSF), a household-scale, intermittently operated slow sand filter. Laboratory and field studies examined Escherichia coli reductions achieved by the BSF. During two laboratory studies, mean E. coli reductions were 94% and they improved over the period of filter use, reaching a maximum of 99%. Field analysis conducted on 55 household filters near Bonao, Dominican Republic averaged E. coli reductions of 93%. E. coli reductions by the BSF in laboratory and field studies were less than those typically observed for traditional slow sand filters (SSFs), although as for SSFs microbial reductions improved over the period of filter use. Further study is needed to determine the factors contributing to microbial reductions in BSFs and why reductions are lower than those of conventional SSFs.     

Development and validation of a FISH-based method for the detection and quantification of E. coli and coliform bacteria in water samples

Monitoring of microbiological contaminants in water supplies requires fast and sensitive methods for the specific detection of indicator organisms or pathogens. We developed a protocol for the simultaneous detection of E. coli and coliform bacteria based on the Fluorescence in situ Hybridization (FISH) technology. This protocol consists of two approaches. The first allows the direct detection of single E. coli and coliform bacterial cells on the filter membranes. The second approach includes incubation of the filter membranes on a nutrient agar plate and subsequent detection of the grown micro-colonies. Both approaches were validated using drinking water samples spiked with pure cultures and naturally contaminated water samples. The effects of heat, chlorine and UV disinfection were also investigated. The micro-colony approach yielded very good results for all samples and conditions tested, and thus can be thoroughly recommended for usage as an alternative method to detect E. coli and coliform bacteria in water samples. However, during this study, some limitations became visible for the single cell approach. The method cannot be applied for water samples which have been disinfected by UV irradiation. In addition, our results indicated that green fluorescent dyes are not suitable to be used with chlorine disinfected samples.     

Coarse filters for pond effluent polishing: comparison of loading rates and grain sizes.

A system comprising a UASB reactor, shallow polishing ponds and shallow coarse filters, treating actual wastewater from the city of Belo Horizonte, Brazil, has been evaluated. The main focus of the research was to compare grain sizes and hydraulic loading rates in the coarse filters. Two filters operating in parallel were investigated, with the following grain sizes: Filter 1: 3 to 10 cm; Filter 2: 8 to 20 cm. Two hydraulic loading rates were tested: 0.5 and 1.0 m3/m3.d. The filter with the lower rock size had a better performance than the filter with the larger rock size in the removal of SS and, as a consequence, BOD and COD. A better performance was obtained with the hydraulic loading rate of 0.5 m3/m3.d, as compared to the rate of 1.0 m3/m3.d. The effluent quality during the period with the lower loading rate was very good for discharge into water bodies or for agricultural reuse (median effluent concentrations from Filter 1: BOD: 20 mg/L; COD: 106 mg/L; SS: 28 mg/L; E. coli: 528 MPN/100 mL).     

Comparison of three different media for the detection of E. coli and coliforms in water.

The European Drinking Water Directive defines reference methods for the enumeration of microbiological parameters in drinking water. The method to be used for Escherichia coli and coliforms is the membrane filtration technique on Lactose TTC agar with Tergitol 7. Many technical drawbacks of the procedure, as well as its limitations regarding the recent taxonomy of coliforms, make it necessary to evaluate alternative methods. Two alternative assays, a chromogenic media (m-ColiBlu24) and a defined substrate technology-DST test (Colilert 18/Quanty Tray) were compared with the ISO standard with attention to the phenotypic characteristic of the isolates. Results showed that the ISO method failed to detect an important percentage of coliforms and E. coli while m-ColiBlu24 and Colilert 18 provided results in a shorter time allowing the simultaneous detection of E. coli and coliforms with no further confirmation steps.     

Field investigation of arsenic in ceramic pot filter-treated drinking water

Ceramic pot filters (CPFs) is one of several household water treatment technologies that is used to treat drinking water in developing areas. The filters have the advantage of being able to be manufactured using primarily locally available materials and local labor. However, naturally-occurring arsenic present in the clay used to make the filters has the potential to contaminate the water in excess of the World Health Organization drinking water standard of 0.01 mg/L. A manufacturing facility in Guatemala routinely rinses filters to reduce arsenic concentrations prior to distribution to consumers. A systemic study was performed to evaluate the change in arsenic concentrations with increasing volumes of rinse water. Arsenic field kit results were compared to standard method laboratory results, and dissolved versus suspended arsenic concentrations in CPF-treated water were evaluated. The results of the study suggest that rinsing is an effective means of mitigating arsenic leached from the filters, and that even in the absence of a formal rinsing program, routine consumer use may result in the rapid decline of arsenic concentrations. More importantly, the results indicate that filter manufacturers should give strong consideration to implementing an arsenic testing program.     

Virus removal in a membrane separation process

Recently, membrane technology has been considered an alternative to conventional water purification. To study the fate of viruses in membrane processes, indigenous coliphages in pilot scale membrane processes located in the eastern part of Tokyo Metropolitan area have been surveyed for 6 months. This plant used river water as its resource and had two microfiltration membrane processes which had different pore sizes (0.2 µm and 0.1 µm) and one ultrafiltration process which had 13,000 nominal molecular weight cut off. To detect indigenous coliphages, E. coli K12 F⁺(A/λ) and E. coli C were used as host bacteria. E. coli K12 F+(A/λ) can detect both DNA and RNA phages and E. coli C can only DNA phage. The resource water contained E. coli K12 phages at 200–1500 PFU/100 mL and the removal ratio of these DNA and RNA phages was lower than that of DNA phage by E. coli C in both MF membrane processes through 6 months. It is thought to be caused by difference of phage size, because DNA phage is bigger than RNA phage in general. The removal ratio of E. coli K12 and E. coli C phages reached 100% in the UF membrane process. According to the comparison of the concentration of phages in solution and eluted from suspended solid in resource and drain, it is thought that most phages concentrated in the drain were absorbed in suspended solids. To make certain of the removal ratio in UF and NF (nanofiltration) processes, high concentrations of coliphage Qβ and poliomyelitis virus vaccine were fed into these processes. The removal ratio of coliphage Qβ in UF and NF processes are 10⁻⁸³ and 10^−6.3 respectively, and the ratio of poliomyelitis virus vaccine in UF and NF are µ10^−6.7 and µ10^−7.3 respectively.     

Zinc-sulphate-heptahydrate coated activated carbon for microbe removal from stormwater

There is a need to develop effective stormwater filters for passive (without any addition of chemicals or energy) and effective removal of pathogens in order to mainstream stormwater harvesting. This study focuses on the development of coated granular activated carbon (GAC) filtration material in order to develop filters for effective removal of pathogens from urban stormwater. Several laboratory trials were performed to gauge the effectiveness of the filters, which use a mixture of the zinc-sulphate-heptahydrate coated GAC and sand, on the removal of Escherichia coli (E. coli) from semi-natural stormwater. On average, a 98% removal of the inflow concentration of E. coli was achieved. Furthermore, there was also an improvement of approximately 25% in the removal of phosphorous. However, it was found that the treated material was leaching zinc. It was important to determine whether the observed removal of E. coli was indirectly caused by the sampling methodology. The results showed that the inactivation of the E. coli in the collected sample was small compared with the inactivation which actually occurred within the filter. This provides much promise to the filter, but the presence of zinc in the outflow demonstrates the need for further investigation into the stabilisation of the coating process.     

Secondary effluent reclamation: combination of pre-treatment and disinfection technologies.

A study was carried out to evaluate the efficiency of secondary effluent additional treatment, using a combination of pre-treatments (ring filter, physico-chemical and infiltration-percolation) followed by disinfection methods (chlorine dioxide, peracetic acid and ultraviolet light). Three different indicator microorganisms were determined: E. coli, total coliforms and somatic bacteriophages. The results show better efficiency of physico-chemical and infiltration-percolation processes. Bacteriophages were eliminated to a lesser extent than bacterial indicators in all the treatment systems. Chlorine dioxide and peracetic acid seems to be more efficient in disinfection than ultraviolet light when a ring filter is the pre-treatment used. For the same doses and contact times, the efficiency of the disinfection methods is higher when the pre-treatment used is the physico-chemical or the infiltration-percolation system. The final effluent quality from the physico-chemical treatment train and the infiltration-percolation treatment train, followed by the disinfectants, achieves an E. coli content that allows the reuse in most of the uses described in the Spanish legislation for wastewater reuse.     

Direct detection of bacterial faecal indicators in water samples using PCR.

The presence of enteric pathogens in water resources represents a serious risk for public health. Therefore, their precise detection, and especially detection of E. coli, which is obviously regarded as the main indicator of faecal contamination of water, is an essential step in ensuring bacterial safety of water. Numerous PCR protocols for detection of E. coli have been published to date. They are usually based on amplification of regions derived from lacZ (beta-D-galactosidase) and uidA (beta-D-glucuronidase) gene sequences. However, these methods are not universal enough for precise detection of all E. coli strains found in water samples. We developed a novel triplex PCR method for detection of E. coli in which cyd gene coding for cytochrome bd complex was co-amplified along with lacZ and uidA genes. Our triplex PCR approach significantly increases the specificity and reliability of E. coli detection in water samples. This approach allowed us to distinguish Shigella flexneri from E. coli. In addition, we were able to detect even non-coliform Klebsiella and Raoutella spp., some of which can also cause infections to humans.     

An antimicrobial polycationic sand filter for water disinfection

A new sand filtration water disinfection technology is developed which relies on the antimicrobial properties of hydrophobic polycations (N-hexylated polyethylenimine) covalently attached to the sand's surface. The efficacy of the filter disinfection process was evaluated both with water spiked with E. coli and with real aqueous effluent from a wastewater treatment plant. For the former, over 7-log reduction in bacterial count was achieved. With real environmental wastewater secondary effluent samples, the E. coli concentration reduction declined to under 2 logs. This reduced inactivation efficiency compared to the model aqueous sample is likely due to the particulate or colloidal matter present that diminishes the contact between the immobilized polycation and the suspended bacteria. Preliminary sand washing methods were tested to assess potential 'regeneration' approaches. Potential advantages of the proposed approach over conventional disinfection in terms of eliminating harmful by-products and reducing energy consumption are discussed.     

Bacteroides spp. as reliable marker of sewage contamination in Hawaii's environmental waters using molecular techniques.

Standard PCR (SPCR) and quantitative PCR (QPCR) assays using primers for general and for human-specific Bacteroides 16S rRNA markers were selected as the molecular tests to assess sewage contamination in recreational waters of Hawaii and these same water samples were assayed for culturable concentrations of selected faecal microbial indicators. The results of this study showed that the general primer for Bacteroides was not useful because ambient and polluted water samples were positive for this marker. However, use of human-specific primers reliably detected sewage contamination. The human-specific Bacteroides detection data supported previously reported conclusions that concentrations of alternative faecal indicators (C. perfringens, FRNA coliphages) but not traditional faecal indicators (faecal coliform, E. coli, enterococci) are reliable indicators of faecal contamination in Hawaii's environmental waters. The QPCR assay for the human-specific Bacteroides 16S rRNA marker was faster, more sensitive and more reliable than comparable SPCR assay because OPCR assay provided additional information such as melting temperatures, which confirmed that the right amplicons were being measured and Ct values, which indicated the relative level of faecal contamination.     

Correlations between silt density index,turbidity and oxidation-reduction potential parameters in seawater reverse osmosis desalination

The reverse osmosis method is one of the most widely used methods of seawater desalination at present.Hydrophilic and desalting membranes in reverse osmosis systems are highly susceptible to the input pollutants.Various contaminants,including suspended organic and inorganic matter,result in membrane fouling and membrane degradation.Fundamental parameters such as the turbidity,the amount of chlorine injection,and silt density index(SDI)are the most predominant parameters of fouling control in the membranes.In this study,the operation system included a water intake unit,a pretreatment system,and an RO system.The pretreatment system encompassed a clarifier,a gravity sand filter,pressurized sand filters,and a cartridge filter.The correlation between the amount of chlorine injection in terms of the oxidation-reduction potential(ORP)and the SDI value of the input water was investigated at a specified site next to the Persian Gulf.The results showed that,at certain intervals of inlet turbidity,injection of a certain amount of chlorine into the raw water has a distinct effect on the decrease of SDI.     

Performance of enviss? stormwater filters: results of a laboratory trial

An experimental study was undertaken by Monash University to develop and test enviss? stormwater treatment and harvesting technologies - non-vegetated filtration systems with an extremely low footprint. This paper focuses on the water quality and hydraulic performance of two systems tested over a 'year' of operation in a Melbourne climate: (1) REUSE enviss? filters, designed for stormwater harvesting systems for non-potable supply substitution, and (2) WSUD enviss? filters, developed to treat urban stormwater prior to discharge to downstream systems. The presence of chlorine as a disinfection agent proved to be very efficient for the removal of microorganisms in REUSE enviss? filters. WSUD enviss? filters had the benefit of providing an elevated nutrient treatment performance, due to an extended depth of filter media. However, nutrient outflow concentrations (total nitrogen (TN) in particular) were found to increase during the testing period. Also, extended dry weather periods were found to have a detrimental effect on the treatment performance of almost all pollutants for both filters (nutrients, Escherichia coli and heavy metals). Although hydraulic conductivity results indicated two or three sediment trap replacements per year are required to maintain filtration rates, it is expected that the compressed loading rate schedule overestimated this maintenance frequency.     

Adsorption kinetics of Escherichia coli and Staphylococcus aureus on single-walled carbon nanotube aggregates

Batch adsorption studies to determine adsorption kinetics of Escherichia coli (E.coli) K12 and Staphylococcus aureus (S.aureus) SH 1000 bacterial cells on single-walled carbon nanotube aggregates were performed at two different initial concentrations. The diffusivity of E. coli cells in single-walled carbon nanotube aggregates obtained was 6.54 x 10(-9) and 8.98 x 10(-9) cm(2)/s, whereas that of S. aureus was between 1.00 x 10(-7) and 1.66 x 10(-7) cm(2)/s respectively. In addition to batch adsorption studies, electron microscopy studies were also conducted. The results suggest that diffusion kinetics of bacterial cells is concentration dependent as well as bacteria dependent. Diffusivity of S. aureus is two orders of magnitude greater than E. coli cells. This proves to be beneficial from an adsorption perspective where it is desired to filter microorganisms (water pretreatment and wastewater post treatment) and from nanotube biosensor perspective where it is desired to simultaneously capture and detect biothreat agents in a shorter span of time.     

Retention of Escherichia coli in municipal sewage by means of planted soil filters in two-stage pilot plant systems.

Different types of constructed wetlands in a pilot-plant system were fed with identical municipal waste water to compare the sanitisation process in two-stage systems. With combinations of a vertical and a horizontal flow filter an E. coli reduction of 5 log10 with an E. coli influent concentration of 10(7) MPN/100 ml was achieved. Using different filter materials in each stage the total performance of the two-stage system was independent from the sequence of these materials. However, using coarser filter material in the first stage makes the filter less prone to clogging and is thus the preferential option with regard to operational reliability.     

Microbiological quality of reclaimed water used for golf courses' irrigation.

Microbial quality of reclaimed water used for irrigation in two golf courses located in the southern Iberian Peninsula (Spain and Portugal) was evaluated. Bacterial indicators for faecal pollution (total and faecal coliforms, Escherichia coli and enterococci) were tested by membrane filtration using appropriate selective media. In addition, somatic E. coli bacteriophages, enteric viruses (entero-, hepatitis A and rota-) and Legionella pneumophila were also analysed. The results obtained showed that all wastewater treatment processes reduced adequately the number of indicator microorganisms although a significant correlation between pathogenic and indicator microorganisms tested was not found. L. pneumophila was detected by PCR but not confirmed by culture. Survival experiments of pathogenic microorganisms in aerosols and irrigated turf are conducted to determine the health hazards for the golf practice and to propose a microbial standard for wastewater used for irrigation of golf courses.     

Assessment of microbial infection risks posed by ingestion of water during domestic water use and full-contact recreation in a mid-southern African region.

A customised Water-related Quantitative Microbial Risk Assessment (WRQMRA) process was used to determine risk of infection to water ingested by users in the south-eastern Free State, South Africa. The WRQMRA consisted of an observed-adverse-effect-level approach (OAELA) and a quantitative microbial risk assessment (QMRA). The OAELA was based on the occurrence of E. coli in the study waters to determine the possible risk of infection and the QMRA probable risk of infection by salmonellae. The WRQMRA was applied to recreational surface resource waters as well as waters from an unprotected spring and waters from the treated municipal supply that were stored in containers for domestic purposes. E. coli numbers were measured against expected infection levels expressed in water quality guidelines, while Salmonella counts were calculated to give the probable infection risk (Pi). Ingestion was based on intake volumes compiled for the various water uses. E. coli occurred in numbers <10(6) in the surface waters, while the untreated spring and treated supply water contained E. coli of <10(2) and <10(1) respectively. Salmonella occurred in numbers of <10(3) in recreational waters, and <10(-1) in water used for domestic purposes. A single exposure to the mean (as well as 95th percentile) risk was calculated using a beta-Poisson dose-response model at ingestion volumes of 100 mL (for full-contact recreation) and 1,318 mL (for domestic water use). Both the OAELA and the QMRA approaches indicated a risk of infection to recreational and domestic water users, even for a single exposure event, with the OAELA either over- or under-estimating the risk of infection for singular exposure events. This indicated that this method, used on its own, could not reliably predict a realistic risk of infection. It is recommended that the full WRQMRA process be used, and further developed to address several uncertainties that became evident during this study.