Competition for growth substrates in river water between Escherichia coli and indigenous bacteria illustrated by high-resolution mass spectrometry.

Escherichia coli normally cannot grow in the environment. One environmental stress that prevents E. coli growth may be the competition for growth substrates with co-existing micro-organisms. In this study, the growth substrates of E. coli were screened by high-resolution mass spectrometry and compared with those of indigenous bacteria in river water. In an incubation experiment, E. coli multiplied in sterilized river water, but did not multiply when indigenous micro-organisms were present in the water. By analysing dissolved organic matter in the river water before and after E. coli growth, 35 compounds were identified as putative growth substrates of E. coli. Among them, 33 compounds were also identified as putative growth substrates of indigenous bacteria. These results indicate that E. coli and indigenous bacteria compete for organic substrates in river water, which could suppress the growth of E. coli.

Ozone disinfection kinetics of poliovirus 1 determined by cell culture assay, RT-qPCR and ethidium monoazide qPCR reduction in a continuous quench-flow reactor.

AIMS: A continuous quench-flow (CQF) reactor was developed to collect samples at the reaction times of less than one second. The reactor is applied to determine ozone disinfection kinetics of poliovirus and to study whether EMA-qPCR can assess the viral infectivity after ozone disinfection. METHODS: Ozone disinfection of poliovirus was conducted in the developed CQF, and the disinfection kinetics were tested in the range of 0·7-5·0 s at ozone concentration of 0·08 and 0·25 mg l-1 . Inactivation, damage on viral genome and damage on capsid integrity were determined by plaque assay, quantitative reverse transcription polymerase chain reaction (RT-qPCR) and ethidium monoazide treatment coupled with RT-qPCR (EMA-qPCR), respectively. RESULTS: By using CQF, 2·18 and 2·76 log10 reductions were observed at the reaction time of 0·7 s and ozone concentration of 0·08 and 0·25 mg l-1 , respectively, followed by tailing. Ozone disinfection kinetics of poliovirus 1 were better fit by the efficiency factor Hom model than by the Chick-Watson model, or the modified Chick-Watson model. Kinetics observed were similar between RT-qPCR and EMA-qPCR assays at the reaction times of <2·0 s and ozone concentrations of 0·08 and 0·25 mg l-1 . At reaction times > 5 s, viral concentration evaluated by EMA-qPCR was reduced in comparison to stable RT-qPCR results. Both assays still underestimated the virus inactivation. CONCLUSION: The simple developed reactor can be used to investigate viral ozone disinfection kinetics and to elucidate inactivation characteristics or mechanisms at very short exposure times. SIGNIFICANCE AND IMPACT OF THE STUDY: The developed CQF reactor is beneficial for better understanding of virus inactivation by ozone, and the reactor can be used to better elucidate disinfection kinetics and mechanisms for future research. This work constitutes an important contribution to the existing knowledge of the application and limitation of the EMA/PMA-qPCR to assess virus infectivity after ozone disinfection.

Identification of estrone-degrading Betaproteobacteria in activated sludge by microautoradiography fluorescent in situ hybridization.

UNLABELLED: Natural oestrogens, which are degraded but not completely removed in wastewater treatment plants, are suspected of causing the endocrine disruption of aquatic organisms in the receiving water body. While several bacterial isolates were reported to be oestrogen-degrading bacteria, our previous study implied that only the unidentified rod-shaped Betaproteobacteria in chains were responsible for estrone (E1) degradation by activated sludge especially at the sub-milligram per litre level. The Betaproteobacteria were suspected to be related to genera Sphaerotilus and Leptothrix according to morphological observations. Probe Spha823 was newly developed to target 16S rRNA gene clones obtained from activated sludge and closely related to the above genera. [(3) H]E1-incubated sludge samples showed that most of the (3) H-labelled cells hybridized with probe Spha823 by microautoradiography (MAR) fluorescent in situ hybridization. Spha823-defined cells were present in all three activated sludge samples tested, where they accounted for up to 3% of the total microbial biomass. Spha823-defined cells comprised 59·5-80·1% of the total MAR-positive cells, which suggested that the Sphaerotilus-Leptothrix-related bacteria were the most abundant micro-organisms involved in E1 degradation (at 200 µg l(-1) ) in the activated sludge samples. SIGNIFICANCE AND IMPACT OF THE STUDY: Estrone (E1) is one of the natural estrogens, which can be degraded but is not always completely removed in wastewater treatment plants. E1 is suspected of causing the endocrine disruption of aquatic organisms in the receiving water body. We identified dominant E1-incorporating bacteria, which should include E1-degrading bacteria, in activated sludge treating domestic wastewater. Sphaerotilus-Leptothrix-related bacteria, which had never been reported in the previous attempts based on culture-dependent approach, occupied 60-80% of the E1-incorporating bacteria. This study demonstrates the identification of functionally active bacteria to degrade micro-pollutants at sub-milligram per litre level.

Model evaluation of faecal contamination in coastal areas affected by urban rivers receiving combined sewer overflows.

Odaiba seaside park is one of the most popular waterfronts in Tokyo Bay, but is easily affected by wet weather pollutant loads through combined sewer overflows (CSOs). The monitoring data of Escherichia coli clearly showed high faecal contamination after a rainfall event on 9-11 November 2007. We estimated the amounts of discharge volume and E. coli pollutant loads of urban rivers receiving CSO from rainfall chambers as well as pumping stations and primary effluent discharge. The result suggested that Sumida River and Meguro River were more influential to the Odaiba coastal area than other sources including the nearest wastewater treatment plant. Subsequently, we simulated the dynamic behaviour of E. coli by a three-dimensional (3D) hydro-dynamic and water quality model. The model simulation reproduced that E. coli concentration after the rainfall event increased rapidly at first and later gradually decreased. The simulations with and without inflow pollutant loads from urban rivers suggested that the E. coli concentration can be influenced by the Meguro River just after the rainfall event and Sumida River about 1 week later. From the spatial and temporal distribution of surface E. coli concentration, after at least 6 days from the rainfall event, high faecal contamination spread to the whole of the coastal area.

Capsid-Damaging Effects of UV Irradiation as Measured by Quantitative PCR Coupled with Ethidium Monoazide Treatment.

The damage to a viral capsid after low-pressure (LP) and medium-pressure (MP) UV irradiation was assessed, using the quantitative or quantitative reverse transcription PCR coupled with ethidium monoazide treatment (EMA-PCR). After UV irradiation, adenovirus 5 (Ad5) and poliovirus 1 (PV1) were subjected to a plaque assay, PCR, and EMA-PCR to investigate the effect of UV irradiation on viral infectivity, genome damage, and capsid damage, respectively. The effectiveness of UV wavelengths in a viral genome and capsid damage of both PV1 and Ad5 was also further investigated using a band-pass filter. It was found that an MPUV lamp was more effective than an LPUV lamp in inactivating Ad5, whereas there was no difference in the case of PV1. The results of viral reduction determined by PCR and EMA-PCR indicated that MP UV irradiation damaged Ad5 capsid. The damage to PV1 and Ad5 capsid was also not observed after LP UV irradiation. The investigation of effects of UV wavelengths suggested that UV wavelengths at 230-245 nm have greater effects on adenovirus capsid in addition to viral genome than UV wavelengths beyond 245 nm.

Characterization of water pollution in drainage networks using continuous monitoring data in the Citadel area of Hue City, Vietnam.

In the Citadel area of Hue City, drainage systems that include canals and ponds are considerable sources of fecal contaminants to inundated water during the rainy season because canals and ponds receive untreated wastewater. It is important to investigate the characteristics of hydraulics and water pollution in canals and ponds. At the canals and ponds, water sampling was conducted during dry and wet weather periods in order to evaluate fecal contamination and to investigate changes in water pollution caused by runoff inflow. Inundated water was also collected from streets during heavy rainfall. At the canals and ponds, concentrations of Escherichia coli and total coliform exceeded the Vietnamese regulation values for surface water in 23 and 24 out of 27 samples (85 and 89%), respectively. The water samples were categorized based on the characteristics of water pollution using cluster analysis. In the rainy season, continuous monitoring was conducted at the canals and ponds using water depth and electrical conductivity (EC) sensors to investigate the dynamic relationship between water level and water pollution. It is suggested that in the canals, high EC meant water stagnation and low EC signified river water inflow. Therefore, EC might be a good indicator of water flow change in canals.

Application of whole sediment toxicity identification evaluation procedures to road dust using a benthic ostracod Heterocypris incongruens.

Road dust is considered to be an important source of sediment contamination in receiving water bodies; however, few studies have evaluated the toxicity of road dust to benthic organisms. This study evaluated the toxicity of road dust to a benthic ostracod, Heterocypris incongruens, using a six-day direct exposure experiment. We applied whole sediment toxicity identification evaluation (TIE) methods to identify the primary group of toxicants in road dust. Three road dust samples from Tokyo caused high ostracod mortality. The addition of hydrophobic adsorbents, Ambersorb and XAD, eliminated toxicity in all samples, suggesting that hydrophobic compounds were the main toxicants in road dust samples. A cation exchange resin, Chelex, also reduced the toxicity of two samples, although the measured concentrations of dissolved heavy metals in the test solution did not exceed the LC(50) values in the literature. In addition, the sum of toxic unit (TU=measured concentration/LC(50)) of each individual metal which predicts the toxicity of the metal mixtures did not exceed 1.0 in all samples, suggesting that heavy metal mixtures did not have additive effects. We hypothesized that the toxicity reduction by XAD and Chelex was due to the removal of hydrophobic compounds, rather than heavy metals. Thus, a toxicity test was conducted on fractions eluted with organic solvents from the XAD and Chelex recovered from one of the road dust samples. Methanol-eluted fractions of XAD and Chelex showed 100 percent ostracod mortality, indicating that the hydrophobic organic compounds removed by these adsorbents were the principal toxicants in the road dust sample.

Runoff load estimation of particulate and dissolved nitrogen in Lake Inba watershed using continuous monitoring data on turbidity and electric conductivity.

Easy-to-measure surrogate parameters for water quality indicators are needed for real time monitoring as well as for generating data for model calibration and validation. In this study, a novel linear regression model for estimating total nitrogen (TN) based on two surrogate parameters is proposed based on evaluation of pollutant loads flowing into a eutrophic lake. Based on their runoff characteristics during wet weather, electric conductivity (EC) and turbidity were selected as surrogates for particulate nitrogen (PN) and dissolved nitrogen (DN), respectively. Strong linear relationships were established between PN and turbidity and DN and EC, and both models subsequently combined for estimation of TN. This model was evaluated by comparison of estimated and observed TN runoff loads during rainfall events. This analysis showed that turbidity and EC are viable surrogates for PN and DN, respectively, and that the linear regression model for TN concentration was successful in estimating TN runoff loads during rainfall events and also under dry weather conditions.

Molecular detection and genotyping of human noroviruses in influent and effluent water at a wastewater treatment plant in Japan.

AIMS: To investigate the prevalence, seasonality and genotype distribution of human noroviruses (NoVs) in wastewater in Japan. METHODS AND RESULTS: Influent and effluent water samples were collected monthly for a year from a wastewater treatment plant and examined for the presence of genogroups I and II (GI and GII) NoVs. Using real-time reverse transcription (RT)-PCR assays, 12 (100%) influent and six (50%) effluent samples were positive for both GI and GII NoV genomes, with a higher prevalence in winter. A total of 152 different NoV strains, comprising 84 GI and 68 GII strains, were identified using seminested RT-PCR assays followed by cloning and sequence analysis. These strains were classified into nine GI genotypes (GI/1, 2, 4, 5, 8, 9, 11, 12 and 14) and 13 GII genotypes (GII/1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 15 and 16), showing considerable genetic diversity. CONCLUSIONS: Based on the partial capsid gene sequences, we identified a great number of NoV strains belonging to many genotypes, demonstrating that genetically diverse NoV strains are co-circulating in aquatic environments and human populations. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results clearly demonstrate the seasonal trend and genetic diversity of NoVs in wastewater.

Washoff behavior of in-sewer deposit in combined sewer system through artificial flushing experiment.

In-sewer deposits in combined sewer systems (CSSs) are closely related with the behavior of first foul flush and combined sewer overflows. However, the research paying attention to the role of in-sewer deposits is quite rare, furthermore, their contributions for the washoff patterns of each pollutant including pathogenic microorganisms are almost never discussed so far. The artificial flushing experiment separating the washoff of in-sewer deposits from the inflow of surface pollutants was carried out to simulate the first foul flush in a CSS. The washoff behaviors of each pollutant including bacterial indicators and enteric viruses were investigated. Several morphological analyses for the concentration and load curves of each parameter were conducted and all patterns were classified according to their washoff characteristics and first foul flush patterns. The washoff behaviors of each pollutant and microorganism are different from each other and categorized into several groups according to their (i) time-series concentration and load curves and (ii) concentration vs. flow rate curves, respectively. The first foul flush patterns of each parameter were to be categorized into typical 3 groups; strong-, partial-, and no first foul flush group. The order of these groups signifies the strength of the first foul flush phenomena and the runoff priority as well.

Characterization of monooxygenase gene diversity in benzene-amended soils.

AIM: To understand soil benzene monooxygenase gene diversity by clone library construction and microarray profiling. METHODS AND RESULTS: A primer set was designed, and benzene monooxygenase gene diversity was characterized in two benzene-amended soils. The dominant sequence types in the clone libraries were distinct between the two soils, and both sequences were assigned to novel clusters. Monooxygenase gene richness and diversity increased after benzene degradation. Oligonucleotide probes for microarray analysis were designed to detect a number of sequenced clones and reported monooxygenase genes. The microarray detected several genes that were not detected in the clone libraries of the same samples. Six probes were detected in more than one soil. CONCLUSIONS: The primer set designed in this study successfully detected diverse benzene monooxygenase genes. The level of diversity may have increased because the degradation of benzene differed from soil to soil. Microarrays have great potential in the comprehensive detection of gene richness as well as the elucidation of key genes for degradation. SIGNIFICANCE AND IMPACT OF THE STUDY: This study introduces a new primer set that may be used to identify diverse benzene monooxygenase genes in the environment; moreover, it demonstrates the potential of microarray technology in the profiling of environmental samples.

Acetate uptake efficiency of polyphosphate-accumulating organisms under exposure to surfactants.

We investigated the influence of surfactants such as linear alkylbenzene sulfonates (LAS) and alcohol ethoxylates (AE) on acetate uptake by polyphosphate-accumulating organisms (PAOs) under anaerobic conditions, using the phosphate requirement for acetate uptake (+DeltaP/-DeltaAc ratio). In order to estimate the +DeltaP/-DeltaAc ratio, anaerobic batch tests were conducted using activated sludge collected from an anaerobic/oxic sequencing batch reactor used to treat municipal wastewater continuously supplemented with a detergent containing LAS and AE. We demonstrated that LAS and AE have both positive and negative impacts on acetate uptake by PAOs. The disadvantage is that long-term exposure to the detergent inhibits acetate uptake by PAOs, thus deteriorating the efficiency, even if the surfactants are no longer present during the tests. Furthermore, the existence of LAS and/or AE with acetate further diminishes the efficiency. The advantage is that LAS and AE are potential sources of polyhydroxyalkanoate for PAOs, because acetate is produced from the surfactants under anaerobic conditions.

Diurnal fluctuation of indicator microorganisms and intestinal viruses in combined sewer system.

Combined sewer overflow (CSO) has been considered to be a source of pathogenic microorganisms for aquatic environment. For the effective control and treatment of CSOs, the microbial behavior in combined sewer system (CSS) needs to be investigated. In this study, whole-day extensive monitoring of indicator microorganisms and intestinal viruses in dry weather flow (DWF) was conducted at a small residential urban drainage area with CSS. All indicator bacteria represented similar diurnal variations in the two different monitoring campaigns; their concentrations gradually decreased to the minimum at the dawn (around 5 a.m.), increased sharply to the maximum around 7 to 8 a.m., and remained rather constant from noon to midnight. On the other hand, neither coliphages nor intestinal viruses showed any concentration peaks in the morning. The maximum/minimum load ratios ranged from 18 to 42 for total coliforms, fecal coliforms and E. coli, whereas those ratios for coliphages, enteroviruses and noroviruses G2 showed greater values than those for indicator bacteria. These results indicate that the diurnal variation patterns of bacterial and viral concentrations in DWF should be considered, which affect the discharge characteristics of each microorganism and the loads of bacteria and viruses in CSOs significantly vary with the overflow time as well.

Competitive adsorption of heavy metals in soil underlying an infiltration facility installed in an urban area.

Accumulation of heavy metals at elevated concentration and potential of considerable amount of the accumulated heavy metals to reach the soil system was observed from earlier studies in soakaways sediments within an infiltration facility in Tokyo, Japan. In order to understand the competitive adsorption behaviour of heavy metals Zn, Ni and Cu in soil, competitive batch adsorption experiments were carried out using single metal and binary metal combinations on soil samples representative of underlying soil and surface soil at the site. Speciation analysis of the adsorbed metals was carried out through BCR sequential extraction method. Among the metals, Cu was not affected by competition while Zn and Ni were affected by competition of coexisting metals. The parameters of fitted 'Freundlich' and 'Langmuir' isotherms indicated more intense competition in underlying soil compared to surface soil for adsorption of Zn and Ni. The speciation of adsorbed metals revealed less selectivity of Zn and Ni to soil organic matter, while dominance of organic bound fraction was observed for Cu, especially in organic rich surface soil. Compared to underlying soil, the surface soil is expected to provide greater adsorption to heavy metals as well as provide greater stability to adsorbed metals, especially for Cu.

Heavy metal (Zn and Cu) complexation and molecular size distribution in wastewater treatment plant effluent.

The size distributions of zinc and copper species in the effluent of a wastewater treatment plant were determined by a combination of ultrafiltration and chelating disk cartridge fractionation. The results showed that 75-87% of total Zn and 84-86% of total Cu were strongly complexed or particle-bound in the final effluents. It was also found that the major part of Cu was bound to ligands in the < 500 Da fraction while the trend for Zn was not so clear and exhibited significant seasonal variability. Labile Cu and Zn were detected not only in the smallest fraction (< 500 Da) but also in the larger fractions. It meant that the labile species in the effluent were not equivalent to free metal ions. Conditional stability constants and ligand concentrations were also determined from the measured metal concentrations by square wave anodic stripping voltammetry. Existence of two types of ligand for each metal was inferred from the experimental data. Conditional stability constant obtained for the stronger type Ligand of Zn was higher than that of Cu, although the estimated Ligand concentrations were almost similar.

Heavy metals speciation in soakaways sediment and evaluation of metal retention properties of surrounding soil.

Heavy metals speciation analysis was carried out on sediment samples accumulated within soakaways in an old stormwater infiltration facility in Tokyo, Japan and on a soil core sample collected near the facility. Heavy metals content in soakaways sediments were much elevated compared to nearby surface soil with the content for Zn, Pb and Cd reaching about 5 to 10 times the content in surface soil. Speciation results revealed that significant amount of the accumulated heavy metals were present in potential mobile fractions, posing threat of release to underlying soil with changing environmental conditions. Detail analyses of soil characteristics indicated significant heterogeneity with depth, especially between the surface soil and underlying soil at site. Decrease in potential adsorption sites with depth was observed in case of underlying soil. Reduced adsorption capacity for heavy metals was evidenced for underlying soil when compared with surface soil. Furthermore, less capability of the soil organic matter to bind heavy metals was evidenced through speciation analyses, which raises concern over the long-term pollution retention potential of the underlying soil receiving infiltrated runoff.

Three dimensional fluorescent spectroscopy analysis for the evaluation of organic matter removal from industrial estate wastewater by stabilization ponds.

The fluorescent excitation emission matrix (FEEM) was utilized to evaluate the removal of organic matter by stabilization ponds, which consist of aeration, facultative, and oxidation ponds of a central wastewater treatment plant of an industrial estate in northern Thailand. The result shows that six fluorescent peaks of influent wastewater and effluent water after aeration, facultative, and oxidation ponds were detected from the FEEMs at 230 nmEx/295 nmEm (A), 275 nmEx/300 nmEm (B), 240 nmEx/355 nmEm (C), 280 nmEx/355 nmEm (D), 275 nmEx/410 nmEm (E) and 330 nmEx/410 nmEm (F). The putative origins of peaks A and B, C and D, and E and F were tyrosine-like, tryptophan-like, and humic and fulvic acid-like substances, respectively. The aeration pond was the main course of action used to reduce the tyrosine-like substances at peaks A and B by 88 and 52%, respectively, and tryptophan-like substances at peaks C and D by 43 and 39%, respectively. There was only a 20 per cent decrease of humic and fulvic acid-like substances at peak E and an 18 per cent decrease at peak F through the use of aeration ponds. Total per cent reductions of total fluorescent organic matter after aeration ponds; facultative ponds; and oxidation ponds were 46, 51 and 54%, respectively. These values were notably similar to the total percent reduction of DOC by the same respective processes at 54, 53, and 55%.

Prolonged deposition of heavy metals in infiltration facilities and its possible threat to groundwater contamination.

A field investigation of infiltration facilities, built two decades ago in Tokyo, was carried out and sediment samples were collected from 12 infiltration inlets of three different locations. Heavy metals contents in the inlet sediment, road dusts and soils samples were analysed and compared. The particle size distribution analysis showed its variation in depth as well as from inlet to inlet. The nature of organic substances present in sediment found changes in particle sizes as well as in depth. The heavy metals content in the sediment samples ranged from 6-143 (Cr), 1-84 (Ni), 49-331 (Cu), 210-2186 (Zn) and 2-332 (Pb) microg/g. The heavy metal content ranges were similar to road dust, which indicated road dust as a possible source for sediment for the infiltration inlets. The lower heavy metals content in many sediment samples than the soil indicated possible release/desorption of heavy metals under newly created environments such as an anaerobic environment. Among the heavy metals there was a relatively good relationship between Cu and Zn, indicating the existence of their common sources.

Transfer of hydrophobic contaminants in urban runoff particles to benthic organisms estimated by an in vitro bioaccessibility test.

An in vitro bioaccessibility test was applied for assessing the transfer of polycyclic aromatic hydrocarbons (PAHs) present in road dust, into benthic organisms living in a receiving water body. The road dust is supposed to be urban runoff particles under wet weather conditions. Sodium dodecyl sulfate (SDS) solution was used as a hypothetical gut fluid. Pyrene, fluoranthene and phenanthrene were the main PAH species in the SDS extractable fraction of road dust, as well as the whole extract. Benzo(ghi)perylene showed relatively low concentrations in the SDS extract in spite of a high concentration in the original dust. The PAH composition in benthic organisms (polychaetes) did not correspond with that of the surrounding sediment and the PAHs detected were also detected in high concentrations in the SDS extract of road dust. When testing the toxicity of the extracted contaminants by a standardised algal toxicity test, SDS extracts of a detention pond sediment showed higher toxicity than the pore water of the corresponding sediment. Sediment suspension showed a comparative toxicity with 0.1% SDS extract. From the results, the in vitro bioaccessibility test seems more suitable to evaluate the exposed contaminants than the traditional organic solvent extraction method and the SDS extracted fraction is applicable to toxicity tests reflecting the digestive process.

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.