Effect of photoreactivation on ultraviolet inactivation of Microcystis aeruginosa.

Microcystis aeruginosa forms algal bloom in lakes. They produce toxic compounds such as microcystin. Against such algal problems, the effect of UV treatment was examined. In UV treatment, the effect of photoreactivation should be examined. Photoreactivation is a repair mechanism of genomic DNA damage by sunlight irradiation. UV treatment causes DNA damages on target cyanobacteria, however sunlight can repair some of these DNA damages. To examine the effect of photoreactivation, both white and yellow light incubations were employed. White light allows both photoreactivation and photosynthesis, while yellow light prohibits photoreactivation and only allows photosynthesis. Microcystis aeruginosa NIES 98 strain and PCC 7806 strain were used as the test cultures. Those cultures were exposed to low-pressure (LP) or medium-pressure (MP) ultraviolet (UV) lamp, then incubated under white or yellow light. Yellow light incubation method was effective to examine photoreactivation. It was revealed that almost six times UV fluence was required to inactivate 99% of Microcystis aeruginosa, under photoreactivation condition, compared with non-photoreactivation condition. Inhibition of photoreactivation could greatly enhance UV treatment efficiency against Microcystis aeruginosa. One of the practical suggestions is to conduct UV treatment at night, when photoreactivation by sunlight rarely takes place. Highly efficient inactivation was achieved by avoiding photoreactivation.

Development of a real-time RT-PCR assay combined with ethidium monoazide treatment for RNA viruses and its application to detect viral RNA after heat exposure.

A method was developed for discriminating damaged viruses or naked viral RNA from intact viruses by ethidium monoazide (EMA) treatment before RT-PCR. The applied EMA treatment consisted of three steps: (1) EMA dose, (2) exposure to light, and (3) additional purification by spin-column gel filtration. Approximately 4-log reduction in viral RNA concentration was observed by adding a dose of 10 µg/mL-EMA with 300 s of light irradiation. Although residual EMA can be an inhibitor of RT-PCR, its effect was reduced by spin-column gel filtration or a QIAamp® Viral RNA Mini Kit. EMA-RT-PCR was applied to the thermally treated PV1. Results of EMA-RT-PCR were similar to the plaque assay when PV1 was thermally inactivated. Although this is a preliminary study investigating applicability of the EMA-RT-PCR method for RNA viruses, the results suggest that the method is potentially applicable for the selective detection of epidemiologically important enteric viruses in water such as enteroviruses and noroviruses.

Detection of genogroup IV norovirus in wastewater and river water in Japan.

AIMS: To test wastewater and river water in Japan for genogroup IV norovirus (GIV NoV). METHODS AND RESULTS: Influent and effluent samples from a wastewater treatment plant and the Tamagawa River water samples were collected monthly for a year. The water samples were concentrated by the adsorption-elution method, using an HA electronegative filter with acid rinse procedure, followed by quantitative detection of GIV NoV using TaqMan-based real-time RT-PCR. Both wastewater and river water samples showed a high positive ratio of GIV NoV during winter and spring. The highest concentration in wastewater and river water was 6.9 x 10(4) and 1.5 x 10(4) copies l(-1), respectively. CONCLUSIONS: Presence of GIV NoV in the environments demonstrates that not only GI and GII NoVs but also GIV strains are circulating and that routine monitoring of GIV NoV in water environments is recommended to understand its epidemics, environmental distribution and potential health risks. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study providing quantitative data on the occurrence of GIV NoV in environmental water over a 1-year period.

Quantitative detection of sapoviruses in wastewater and river water in Japan.

AIMS: To detect sapoviruses at a wastewater treatment plant (WWTP) and in a river in Japan, quantitatively. METHODS AND RESULTS: Influent and effluent samples at a WWTP and river water samples were collected monthly for 1 year. The water samples were subjected to virus concentration using an HA electronegative filter, followed by quantification of sapoviruses using real-time PCR. The concentration of sapoviruses in influent ranged from 2.8 x 10(3) to 1.3 x 10(5) copies per litre, showing a higher value in winter. Seven (58%) of 12 effluent samples were positive for sapoviruses, as were 23 (64%) of 36 river water samples collected from three sites along the Tamagawa River. CONCLUSIONS: Sapoviruses were abundant in the influent even in the nonepidemic period, suggesting that sporadic and asymptomatic infections occur throughout the year. Increasing concentration of sapoviruses was discharged into the river during the epidemic period winter. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study demonstrating the quantitative detection of sapoviruses in aquatic environments.

Quantitative analysis of human enteric adenoviruses in aquatic environments.

AIMS: The aim of this study was to determine human adenoviruses (HuAdVs) in aquatic environments by real-time polymerase chain reaction (PCR). METHODS AND RESULTS: In order to describe the ratio of enteric serotypes to the total HuAdVs, the primer set specific for the enteric serotypes 40 and 41 was used in parallel with the universal primer set for all 51 serotypes of HuAdVs. The enteric serotypes of HuAdVs were detected at the concentration of 7.3-1500 PCR-detection units (PDU) per ml in raw sewage (n = 17), 0.00060-4.1 PDU ml(-1) in secondary-treated sewage before chlorination (n = 17), 0.0018-7.0 PDU ml(-1) in river water (n = 36), and 0.032-6.1 PDU ml(-1) in seawater (n = 18). The concentration of HuAdVs, determined by the universal primer set, was equivalent to that of enteric serotypes in almost all the samples tested. CONCLUSIONS: Enteric serotypes were predominant among all serotypes of HuAdVs in the aquatic environments. SIGNIFICANCE AND IMPACT OF THE STUDY: The abundance of enteric serotypes of HuAdVs should be more emphasized than other serotypes in order to assess the risk of their infection via water.

Accumulation of copper and silver onto cell body and its effect on the inactivation of Pseudomonas aeruginosa.

Pseudomonas aeruginosa, a gram-negative rod bacterium, is a causative agent of waterborne pneumonia and presents high tolerance against conventional disinfectants. The inorganic biocidal reagents, copper and silver, were applied to inactivate P. aeruginosa inoculated in a synthetic drinking water (SDW). Additionally, the relationship of the specific amount of accumulated copper and silver reagents (Cs) on P. aeruginosa with inactivation profile was elucidated in this study. Flow cytometry (FCM) following staining with SYTO 9 and PI was used for detection of bacterial viability and density. Individual copper and silver reagents, and their combination, exhibited excellent biocidal abilities even at the concentration of 0.05 mgCu/L and 0.005 mgAg/L. The critical amounts of accumulated disinfectant (Cs) were calculated at 2.82 x 10(-7) microgCu/cells and 5.13 x 10(-8) microgAg/cells; at an incubation of 70 h. Consequently, the role of disinfectant on the inactivation of P. aeruginosa and the assessment of biocidal ability of copper, silver, and their combination were successfully explained by evaluating the terms Cs and Cc.

Quantification and genotyping of Cryptosporidium spp. in river water by quenching probe PCR and denaturing gradient gel electrophoresis.

A new detection method was developed for the simultaneous quantification and genotyping of Cryptosporidium spp. in river water. Several modifications made to the US EPA Method 1623 enabled high and stable recovery of Cryptosporidium from 40 L of river water (geometric mean = 35%, standard deviation = 8.7%). Quenching probe PCR (QProbe PCR) was used to quantify the 18S rRNA gene of Cryptosporidium spp. This method could successfully detect single oocysts in a sample, and the lower quantitation limit was as low as 2.5 oocysts/sample. In addition, denaturing gradient gel electrophoresis (DGGE) followed by DNA sequencing was used to identify the genotypes. These methods were applied to detect Cryptosporidium spp. in the Koyama River, Japan. The positive ratio was 69% (11/16) with the maximum concentration of 59 oocysts/100 L. Seven genotypes including two novel ones were identified. These results showed that this detection method could provide valuable information on Cryptosporidium in river water, both in the concentration and in the genotypes, which is essential for the precise assessment of waterborne risk to human health.

Monitoring of human enteric viruses and coliform bacteria in waters after urban flood in Jakarta, Indonesia.

Floodwaters in Kampung Melayu village, Jakarta, Indonesia, as well as river water and consumable water (including groundwater and tap water) samples in flooded and non-flooded areas, were quantitatively analysed to assess occurrence of viruses and total coliforms and E. coli as bacterial indicators after flooding event. High numbers of enterovirus, hepatitis A virus, norovirus (G1, G2) and adenovirus were detected at high concentration in floodwaters and waters sampled from Ciliwung River which runs across metropolitan Jakarta and is used widely for agriculture and domestic purposes by poor residents. One out of three groundwater wells in the flooded area was contaminated with all viruses tested while no viruses were found in groundwater samples in non-flooded areas and tap water samples. The results revealed that human enteric viruses, especially hepatitis A virus and adenovirus, were prevalent in Jakarta, Indonesia. This study suggested that flooding posed a higher risk of viral infection to the people through contamination of drinking water sources or direct contact with floodwaters.

Effects of rainfall on the occurrence of human adenoviruses, total coliforms, and Escherichia coli in seawater.

A two-month survey was conducted in order to evaluate the effects of rainfall on the fate of microorganisms in seawater in the Tokyo Bay, Japan. The seawater sample (1,000 mL) was applied to a method to concentrate virus, followed by a quantification of human adenoviruses using the real-time PCR. Total coliforms and E. coli, which were determined by the colony forming method, were detected in all 47 seawater samples, while human adenoviruses were detected in 38 (81%) of the samples. The concentration of tested microorganisms showed 1-2 log units increase after rainfall events, followed by the gradual decrease to the level before the rainfall within a few days.

Seasonal profiles of human noroviruses and indicator bacteria in a wastewater treatment plant in Tokyo, Japan.

The seasonal profiles of microorganisms in raw sewage, secondary-treated sewage, and final effluent at a wastewater treatment plant in Tokyo, Japan, were quantitatively determined each month for one year, from July 2003 to June 2004. Human noroviruses, which were determined by real-time PCR, in raw sewage varied from 0.17-260 copies/mL for genotype 1 and from 2.4-1900 copies/mL for genotype 2, showing much higher values in winter, the epidemic season. The concentration of total coliforms, Escherichia coli, or F-specific phages in raw sewage was almost constant throughout the year. Human noroviruses of genotype 2 were removed most effectively (3.69 log10 on average) at the wastewater treatment plant, followed by E. coli (3.37 log10), total coliforms (3.05 loglo), F-specific phages (2.81 log10), and human noroviruses of genotype 1 (2.27 log10). The removal ratio of human noroviruses was almost constant, independent of the initial concentration of the viruses in raw sewage, which led to the increasing concentration of human noroviruses in final effluent in winter. None of the tested bacteria was judged to be a reliable indicator of human noroviruses in final effluent.

Factors causing PAC cake fouling in PAC-MF (powdered activated carbon-microfiltration) water treatment systems.

Two pilot-scale powdered activated carbon-microfiltration (PAC-MF) reactors were operated using river water pretreated by a biofilter. A high permeate flux (4 m/d) was maintained in two reactors with different particle sizes of PAC. High concentration (20 g/L) in the PAC adsorption zone demonstrated 60-80% of organic removal rates. Analysis on the PAC cake fouling demonstrated that attached metal ions play more important role than organic matter attached on PAC to the increase of PAC cake resistance. Effects of factors which may cause PAC cake fouling in PAC-MF process were investigated and evaluated by batch experiments, further revealing that small particulates and metal ions in raw water impose prominent influence on the PAC cake layer formation. Fe (II) precipitates after being oxidized to Fe (III) during PAC adsorption and thus Fe(ll) colloids display more significant effect than other metal ions. At a high flux, PAC cake layer demonstrated a higher resistance with larger PAC due to association among colloids, metals and PAC particles, and easy migration of small particles in raw water into the void space in the PAC cake layer. Larger PAC possesses much more non-uniform particle size distribution and larger void space, making it easier for small colloids to migrate into the voids and for metal ions to associate with PAC particles by bridge effect, hence speeding up and intensifying the of PAC cake fouling on membrane surface.

Repressive effects of yeast extract on photoreactivation of Escherichia coli.

Photoreactivation of Escherichia coli K12 (IFO 3301), in the presence or absence of yeast extract (YE), was investigated after inactivation by low-pressure UV lamp. An endonuclease sensitive site (ESS) assay was used to determine the UV-induced pyrimidine dimers in the genome of E. coli, while a colony-forming ability (CFA) test was also used to examine the survival ratio of E. coli. The YE solution reduced the CFA recovery at a final concentration of 125 mg/L. A dialysis of the YE solution indicated that the YE fraction (with nominal molecular weight >1,000 and <3,500) was effective at repressing the CFA recovery. Interestingly, the repair of ESS was equivalent regardless of the presence of the YE dialysate, while the CFA recovery was significantly repressed in the presence of YE. It was, therefore, suggested that YE components, probably with molecular weights of 1,000-3,500, were effective at repressing the CFA recovery of E. coli without affecting the ESS repair during photoreactivation.

Series of surveys for enteric viruses and indicator organisms in Tokyo Bay after an event of combined sewer overflow.

Combined sewer overflows (CSOs) have been recognised as one of the serious sources of pollution to the water environment during rain events, although field surveys to investigate the effect of their magnitude and duration on receiving waters have been very limited. The fates of enteric viruses (norovirus G1, G2, enteroviruses) and coliforms were determined in the wastewater treatment plant on a fine day and on a rainy day. Not all microorganisms were reduced in the primary treatment, but were reduced in the secondary treatment. Occurrences of enteric viruses and levels of coliforms were surveyed in the receiving coastal area after a CSO event, with the profiles of the enteric viruses in the coastal seawater being almost at the same positive ratio for 4 d after the CSO event.

Cryptosporidium monitoring system at a water treatment plant, based on waterborne risk assessment.

The water volume required for daily monitoring of Cryptosporidium (which can statistically ensure an annual risk of infection below 10(-4)), was assessed by evaluating the applicability of the Poisson lognormal (PLN) distribution in microbial risk assessment. PLN showed as good a fit to the observed data as to the negative binomial distribution. From the estimated PLN distributions for the source and finished water, the efficacy of the oocyst removal by the conventional water treatment process was estimated to follow log-normal distribution (median = 3.16 log10, 95% CI = 4.27-2.05 log10). The 365 consecutive negative results of daily monitoring for 180 L of finished water were found to be statistically equivalent to the annual risk of infection below 10(-4). This research also suggested the possibility of applying a qualitative detection method, such as CC-PCR, as a routine monitoring method for the quantitative risk management.

Inactivation differences of microorganisms by low pressure UV and pulsed xenon lamps.

UV disinfection has been applied to water treatment in recent years with low-pressure and medium-pressure UV lamps mainly used as the light source. In general, UV disinfection is considered to be inefficient with water of high turbidity because of inhibition of light penetration. Additionally, photoreactivation may be a problem that should be considered in case a disinfected water is discharged to the environment where sunlight causes reactivation. Recently, other types of lamps have been proposed including a flush-type lamp (such as a pulsed-xenon lamp) that emits high energy and wide wavelength intermittently. In this study, the difference between inactivation efficiencies by low-pressure UV (LPUV) and pulsed-xenon (PXe) lamps was investigated using two coliphage types and three strains of Escherichia coli. PXe had a suppressive effect on photoreactivation rate of the E. coli strains even though there was no significant effect on inactivation rate and maximum survival ratio after photoreactivation. PXe also had a benefit when applied to high turbidity waters as no tailing phenomena were observed in the low survival ratio area although it was observed in LPUV inactivation. This efficiency difference was considered to be due to the difference in irradiated wavelength of both lamps.

Role of hydrogen peroxide and hydroxyl radical in producing the residual effect of ultraviolet radiation.

Residual effect of UV radiation on Microcystis aeruginosa and Escherichia coli K12 A/lambda (F+) was studied under various conditions to examine if the formation of hydrogen peroxide (H2O2) and hydroxyl radical (OH.) can explain the residual biocidal action of UV radiation. Survival of test organisms, that is, M. aeruginosa and E. coli K12 A/lambda (F+), in UV-irradiated synthetic water was monitored to assess the residual effect after the UV radiation. Synthetic water with various compositions was used to understand the role of transition metals and photosensitizing organic molecules in producing the residual biocidal action. Ultraviolet-irradiated synthetic water containing a photosensitizing element and ferric ion (Fe3+) showed residual biocidal effect for more than 7 days. No residual effect was observed in the absence of any photosensitizing element. A close relationship was observed between the residual effect and the formation of hydrogen peroxide in the UV-irradiated water. Scavenging of hydrogen peroxide and hydroxyl radical from the UV-irradiated water significantly reduced the residual biocidal action. Ultraviolet-irradiation of synthetic water containing ferric ion was found to promote the formation of ferrous ion (Fe2+). Because ferrous ion can produce reactive species in the presence of hydrogen peroxide, transition metals and hydrogen peroxide are believed to play a key role in producing the residual effect of UV radiation through the formation of reactive species, for example, hydroxyl radical.

Assessment of risk of infection due to Cryptosporidium parvum in drinking water.

The risk of infection of Cryptosporidium via drinking water was assessed using Monte Carlo simulation with the field survey data of the Sagami River watershed. The levels of Cryptosporidium in this river were found to follow the lognormal distribution. From the counted data, the median level of the Miyayama sampling point was estimated to be 5.7 oocysts per 100L. To calculate the annual risk of infection due to Cryptosporidium in drinking water, the Cryptosporidium level of Miyayama sampling point was used as the water source of the waterworks. The 95% percentile of the annual risk of infection was found to be 10(-2.60). If the daily risk was eliminated when the level of Cryptosporidium in treated water exceeded 1 oocyst per 20L, the 95% percentile of the annual risk was reduced by about 1 log. To reduce the 95% value of the annual risk lower than 10(-4), the risk of days with levels of Cryptosporidium in treated water exceeding 1 oocyst per 80L should be eliminated.

Rainwater drainage management for urban development based on public-private partnership.

The Urban Development Corporation (UDC) is one of the biggest implementation bodies for urban development in Japan. UDC has developed rainwater infiltration technology since 1975. This technology has effectively reduced runoff to a river and sewer system in the new town project areas. Recently, UDC has developed a new system which is defined as a "Rainwater Recycle Sewer System", which is supported by "Rainwater Storage and Infiltration Technology (RSIT)" applicable to new town creation and urban renewal. The new system consists of two elements: RSIT components based on Public-Private Partnership (PPP) and a stormwater drainage system. Herein, the private sector is responsible for the main part of RSIT, and the public sector is responsible for the stormwater drainage from the development area. As a result, the capacity of public facilities, such as rainwater sewers and stormwater reservoirs, can be reduced effectively. In parallel, the initial/running cost of public facilities is expected to be reduced. In conclusion, the authors would stress the importance of a co-maintenance system also based on PPP, which will be required especially in order to properly operate the whole system for the long term.