Ikuro Kasuga

Evaluation of microbial regrowth potential by assimilable organic carbon in various reclaimed water and distribution systems

Microbial regrowth introduces several problems to the use of reclaimed water, such as health concerns, aesthetic deterioration, and biofouling. This study evaluated assimilable organic carbon (AOC), which is a part of the biodegradable organic matter promoting microbial growth, in water reclamation systems in Japan. The AOC concentration in the reclaimed water from various treatment processes ranged between 36 and 446 μg C/L (median 316 μg C/L). The AOC concentration in the reclaimed water from the plants equipped with ozonation was significantly higher - more than two times - than that in the reclaimed water from plants equipped with other processes. UV and chlorine also changed the AOC concentration slightly. Moreover, reverse osmosis produced reclaimed water with the lowest AOC content. Processes equipped with membrane filtration were effective in removing seed microorganisms that enter the distribution system. Microbial growth in reclaimed water distribution systems occurred when the total and free residual chlorine was lower than 0.36 and 0.09 mg/L, respectively. The AOC reduction occurred simultaneously with regrowth, which suggests that AOC could support microbial growth in reclaimed-water-distribution systems. As the residual chlorine is often depleted during distribution and storage, it is essential to control the AOC to suppress microbial growth.

Predominance of ammonia-oxidizing archaea on granular activated carbon used in a full-scale advanced drinking water treatment plant.

Ozonation followed by granular activated carbon (GAC) is one of the advanced drinking water treatments. During GAC treatment, ammonia can be oxidized by ammonia-oxidizing microorganisms associated with GAC. However, there is little information on the abundance and diversity of ammonia-oxidizing microorganisms on GAC. In this study, the nitrification activity of GAC and the settlement of ammonia-oxidizing archaea (AOA) and bacteria (AOB) in GAC were monitored at a new full-scale advanced drinking water treatment plant in Japan for 1 year after plant start-up. Prechlorination was implemented at the receiving well for the first 10 months of operation to treat ammonia in raw water. During this prechlorination period, levels of both AOA and AOB associated with GAC were below the quantification limit. After prechlorination was stopped, 10(5) copies g-dry(-1) of AOA amoA genes were detected within 3 weeks and the quantities ultimately reached 10(6)-10(7) copies g-dry(-1), while levels of AOB amoA genes still remained below the quantification limit. This observation indicates that AOA can settle in GAC rapidly without prechlorination. The nitrification activity of GAC increased concurrently with the settlement of AOA after prechlorination was stopped. Estimation of in situ cell-specific ammonia-oxidation activity for AOA on the assumption that only AOA and AOB determined can contribute to nitrification suggests that AOA may account for most of the ammonia-oxidation. However, further validation on AOB contribution is required.

Sorption behavior of heavy metal species by soakaway sediment receiving urban road runoff from residential and heavily trafficked areas.

Groundwater contamination by heavy metals from infiltration facilities receiving road runoff is of potential concern. In this study, sorption tests were conducted to evaluate the influence of the water quality of road runoff, especially dissolved organic matter (DOM), on the sorption of heavy metal species by soakaway sediment. Sequential batch tests were conducted to assess metal sorption by the soakaway sediment receiving road runoff from residential and heavily trafficked areas. Ni was adsorbed by the sediment, indicating that soakaway sediments function to prevent groundwater contamination by Ni. In contrast, Zn was released from the soakaway sediment in sorption tests using heavily trafficked road dust leachates. Ni, Cu, Zn, and dissolved organic carbon concentrations were higher in soakaway sediment leachates obtained by sorption tests using heavily trafficked road dust leachates than those using residential road dust leachates, suggesting traffic activities contaminate these pollutants. A large portion of Zn, released from the soakaway sediment, existed as stable complexes. DOM in road runoff possibly enhances the release of Zn from the sediments within infiltration facilities and might cause groundwater contamination.

Potential mobility of heavy metals through coupled application of sequential extraction and isotopic exchange: Comparison of leaching tests applied to soil and soakaway sediment

Artificial infiltration facilities (AIFs) that enhance groundwater recharge and regulate urban runoff are going to be an integral element of the urban infrastructure. However, AIFs provide a sink which trap pollutants that are likely to cause groundwater contamination. The current study aimed first to examine the mobility characteristics of Cu, Zn, and Pb through soil and soakaway sediment using an integrated analytical approach for column leaching with artificial road runoff (ARR) and then to differentiate the sorption patterns among different samples, (i.e., surface soil, underlying soil, and soakaway sediments) using mass balances. In addition, the study compares metal retention and release under continuous and intermittent flow conditions. Column leaching experiments were conducted using batches for 10 and 30 d under continuous flow condition and for 20 d of intermittent leaching. Heavy metal content and partitioning in soil and sediment used in columns were well characterized before and after leaching experiments. The results showed that a gradual increase in pH and decrease in dissolved organic carbon had pronounced effect on the mobilization of heavy metals. Pb showed the highest retention compared to Cu and Zn which implies that metal complexes play a pivotal role in metal transport. Labile fractions were found to be trapped by the solid materials for retention and their high concentration in ARR is a major concern from the pollution point of view through infiltration facilities. Results obtained in this study predict the risk associated with the release of retained heavy metal under changing environmental conditions in AIFs.

Effects of rainfall events on the occurrence and detection efficiency of viruses in river water impacted by combined sewer overflows

Rainfall events can introduce large amount of microbial contaminants including human enteric viruses into surface water by intermittent discharges from combined sewer overflows (CSOs). The present study aimed to investigate the effect of rainfall events on viral loads in surface waters impacted by CSO and the reliability of molecular methods for detection of enteric viruses. The reliability of virus detection in the samples was assessed by using process controls for virus concentration, nucleic acid extraction and reverse transcription (RT)-quantitative PCR (qPCR) steps, which allowed accurate estimation of virus detection efficiencies. Recovery efficiencies of poliovirus in river water samples collected during rainfall events (<10%) were lower than those during dry weather conditions (>10%). The log10-transformed virus concentration efficiency was negatively correlated with suspended solid concentration (r(2)=0.86) that increased significantly during rainfall events. Efficiencies of DNA extraction and qPCR steps determined with adenovirus type 5 and a primer sharing control, respectively, were lower in dry weather. However, no clear relationship was observed between organic water quality parameters and efficiencies of these two steps. Observed concentrations of indigenous enteric adenoviruses, GII-noroviruses, enteroviruses, and Aichi viruses increased during rainfall events even though the virus concentration efficiency was presumed to be lower than in dry weather. The present study highlights the importance of using appropriate process controls to evaluate accurately the concentration of water borne enteric viruses in natural waters impacted by wastewater discharge, stormwater, and CSOs.

Analysis of the phylogenetic diversity of estrone-degrading bacteria in activated sewage sludge using microautoradiography–fluorescence in situ hybridization

In situ uptake of [2,4,6,7-3H(N)]estrone ([3H]E1) by the major phylogenetic groups present in activated sludge samples from two different municipal wastewater treatment plants was investigated using microautoradiography-fluorescence in situ hybridization (MAR-FISH). Approximately 1-2% of the total cells confined in the samples by an EUB probe mix contributed to E1 assimilation. Almost all the detected E1-assimilating cells involved in the early phase of E1 degradation were affiliated with the Beta- and Gammaproteobacteria. In the early phase of E1 degradation, no E1-assimilating cells affiliated with the Alphaproteobacteria, Actinobacteria, the Cytophaga-Flavobacterium cluster of phylum Bacteroidetes, or the phyla Chloroflexi, Nitrospira and Planctomycetes were detected. Bacteria affiliated with the Betaproteobacteria in the shape of long rods or chains of rods were found to contribute most to in situ E1 degradation. They contributed 61% and 82% of total E1-assimilating cells in cultures from two sources of activated sludge spiked with [3H]E1. The E1-degrading bacteria related to the Betaproteobacteria differed phylogenetically from the aerobic E1-degrading bacterial isolates reported in previous studies. In addition, MAR-FISH revealed the significant contribution of E1-degrading bacteria affiliated with the Gammaproteobacteria in the degradation of E1 in activated sludge.

Evaluation of autotrophic growth of ammonia-oxidizers associated with granular activated carbon used for drinking water purification by DNA-stable isotope probing

Nitrification is an important biological function of granular activated carbon (GAC) used in advanced drinking water purification processes. Newly discovered ammonia-oxidizing archaea (AOA) have challenged the traditional understanding of ammonia oxidation, which considered ammonia-oxidizing bacteria (AOB) as the sole ammonia-oxidizers. Previous studies demonstrated the predominance of AOA on GAC, but the contributions of AOA and AOB to ammonia oxidation remain unclear. In the present study, DNA-stable isotope probing (DNA-SIP) was used to investigate the autotrophic growth of AOA and AOB associated with GAC at two different ammonium concentrations (0.14 mg N/L and 1.4 mg N/L). GAC samples collected from three full-scale drinking water purification plants in Tokyo, Japan, had different abundance of AOA and AOB. These samples were fed continuously with ammonium and (13)C-bicarbonate for 14 days. The DNA-SIP analysis demonstrated that only AOA assimilated (13)C-bicarbonate at low ammonium concentration, whereas AOA and AOB exhibited autotrophic growth at high ammonium concentration. This indicates that a lower ammonium concentration is preferable for AOA growth. Since AOA could not grow without ammonium, their autotrophic growth was coupled with ammonia oxidation. Overall, our results point towards an important role of AOA in nitrification in GAC filters treating low concentration of ammonium.

Concentration-dependent response of estrone-degrading bacterial community in activated sludge analyzed by microautoradiography-fluorescence in situ hybridization

Inefficient removal of estrone (E1) in wastewater treatment plants (WWTPs) causes feminizing effects in male aquatic creatures. As E1 is mainly removed by biodegradation, investigation of E1 degradation is important to determine better removal strategies. Using microautoradiography-fluorescence in situ hybridization (MAR-FISH), we demonstrated that the structures of [(3)H]E1-incorporating bacterial communities were different at different E1 concentrations applied to activated sludge. At 200 μg/L E1, almost all [(3)H]E1-incorporating cells were associated with either Betaproteobacteria or Gammaproteobacteria (60% and 40% of MAR (+) cells, respectively). The proportion of Betaproteobacteria and Gammaproteobacteria in the total number of [(3)H]E1-incorporating cells decreased as the concentration of E1 decreased. In contrast, the proportion of Alphaproteobacteria in the total number of [(3)H]E1-incorporating cells increased as the concentrations of E1 decreased. At the lowest applied concentration (540 ng/L), almost all the [(3)H]E1-incorporating cells were Alphaproteobacteria (96%). The results of MAR-FISH applied to sludge samples collected from various plant locations and activated sludge processes, and during different seasons also demonstrated the high contribution of Alphaproteobacteria to the entire E1-degrading bacterial community (50.4 ± 11% of the total number of [(3)H]E1-incorporating cells) at 1 μg/L E1. Since the E1 concentration in domestic wastewater is at sub-μg/L levels, the key E1 degraders in activated sludge of domestic WWTPs are probably be Alphaproteobacteria. All [(3)H]E1-incorporating Alphaproteobacteria were hybridized with probe ALF968. Few MAR (+) cells were Sphingomonadales. An E1-degrading bacterial community at low E1 concentration appeared to consist of diverse bacterial groups of Alphaproteobacteria. This study suggested that substrate concentration is an essential factor for revealing E1-degrading bacteria in complex communities.

Time-Resolved DNA Stable Isotope Probing Links Desulfobacterales- and Coriobacteriaceae-Related Bacteria to Anaerobic Degradation of Benzene under Methanogenic Conditions

To identify the microorganisms involved in benzene degradation, DNA-stable isotope probing (SIP) with 13C-benzene was applied to a methanogenic benzene-degrading enrichment culture. Pyrosequencing of ribosomal RNA (rRNA) gene sequences revealed that the community structure was highly complex in spite of a 3-year incubation only with benzene. The culture degraded 98% of approximately 1 mM 13C-benzene and mineralized 72% of that within 63 d. The terminal restriction fragment length polymorphism (T-RFLP) profiles of the buoyant density fractions revealed the incorporation of 13C into two phylotypes after 64 d. These two phylotypes were determined to be Desulfobacterales- and Coriobacteriaceae-related bacteria by cloning and sequencing of the 16S rRNA gene in the 13C-labeled DNA abundant fraction. Comparative pyrosequencing analysis of the buoyant density fractions of 12C- and 13C-labeled samples indicated the incorporation of 13C into three bacterial and one archaeal OTUs related to Desulfobacterales, Coriobacteriales, Rhodocyclaceae, and Methanosarcinales. The first two OTUs included the bacteria detected by T-RFLP-cloning-sequencing analysis. Furthermore, time-resolved SIP analysis confirmed that the activity of all these microbes appeared at the earliest stage of degradation. In this methanogenic culture, Desulfobacterales- and Coriobacteriaceae-related bacteria were most likely to be the major benzene degraders.

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.