In situ spatial organization of ammonia-oxidizing bacteria in wastewater biofilms was investigated using fluorescent in situ hybridization (FISH) with 16S rRNA-targeted oligonucleotide probes in combination with confocal scanning laser microscopy (CSLM) and digital image analysis. In situ hybridization of steady-state biofilm samples indicated more than 65% of ammonia-oxidizing bacteria present in the biofilm formed dense spherical clusters with the diameter of less than 10 μm and 10∼20% formed clusters with the diameter of more than 10 μm. Most of ammonia-oxidizing bacteria were present in states of small clusters (d<5 μm) in the surface biofilm, because the bigger clusters were imposed on the higher shear stress and consequently sloughed off.
On the other hand, about 4∼10 times higher bacterial population were found as the bigger clusters (d=5∼20 μm) in the bottom biofilm than in the surface, creating a spatial gradient of ammonia-oxidizing bacteria throughout the biofilm. Furthermore, evaluation of series of optical sections through the clusters taken by the CSLM showed that a cluster with the diameter of approximately 25 μm consisted of up to 9,000 rod-shaped cells. Areal cell density within the clusters was found to be in the range of 0.9∼1.1 cells·μm^-2 with areal fraction of cells of 51 ± 6%, indicating that these clusters have relatively large openings. These experimental results suggested that the interior biofilm structure was porous and heterogeneous, the spatial distribution of ammonia-oxidizing bacteria was strongly influenced by the hydraulic shear stress and substrate concentration profiles within the biofilm.

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In this study, we focused on the ozone/hydrogen peroxide water treatment system, one of the advanced oxidation processes, which is expected to be applicable to the treatment of various kinds of waste water. We experimentally investigated the effect of operational parameters with a reaction system with single ozone contactor, and tried to determine the appropriate conditions for the high performance of the system. As the results of this research, we found the existence of the optimal value of overall volumetric mass transfer coefficient, the necessity of maintaining the concentrations of dissolved ozone and hydrogen peroxide in an appropriate range, and the importance of controlling the ozone dose for the high performance of the system. These results also suggested that multiple injection of ozone and hydrogen peroxide is effective fir the high performance. Thus, we have experimentally shown that the multiple injection system achieves higher TOC removal than the single injection system at the same doses of ozone and hydrogen peroxide.

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Ammonium oxidation in wastewater biofilms grown on rotating biological contactors (RBCs) was investigated by using microelectrodes and fluorescent in situ hybridization (FISH) technique. The density of ammonia-oxidizers which formed dense cell cluster was about 5 times higher in the innermost biofilm than in the middle and surface of the biofilm. Due to the presence of ammonia-oxidizers throughout the biofilm, ammonium oxidation zone was expanded with increasing in oxygen penetration depth, which consequently resulted in an increase of the overall ammonium flux into the biofilm. Specific ammonium oxidation rate was dependent upon the density of ammonia-oxidizers. Although more ammonia-oxidizers were detected in the innermost layer, they might be in the dormant state and not contribute ammonium oxidation during the RBC operation because of substrate transport limitation. Furthermore, the fraction of O₂ consumption for nitrification to total O₂ consumption was in the range of 35% -53% in a medium with ammonium as the sole electron donor, indicating the biofilm consumed a significant amount of oxygen for other than nitrification. It implied that heterotrophs would grow and outcompete nitrifiers in biofilms even though there was no organic carbon source in the medium. In conclusion, the combination of microelectrode measurements and FISH technique made it possible to relate the spatial organization of ammonia-oxidizers with their in situ ammonium oxidation rates in biofilms, which obviously providing more insight into the nitrification process in wastewater biofilm systems.

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Immobilized Alcaligenes faecalis, a typical heterotrophic nitrifier, was added into activated sludge to make A. faecalis predominant genus in the activated sludge. Responses of the systems to variation of DO, pH and quantities of inoculated A. faecalis were tested and behaviors of the systems were evaluated. Experimental results showed that: the systems containing A. faecalis generally produced N₂O 5 times less than those containing only activated sludge. In addition, the systems containing A. faecalis had a higher total nitrogen removal capability, average total nitrogen removal efficiencies of them were 10% higher than those of the activated sludge systems. Little influences appeared to TOC removal after the addition of immobilized A. faecalis. Among the experimental conditions, low DO concentration, high amount of A. faecalis and neutral pH led to low N₂O emission and high total nitrogen removal. Therefore, increasing quantity of A. faecalis in activated sludge should be encouraged for controlling N₂O emission and removing nitrogen in wastewater treatment. N₂O producing ways were put forth to explain the influences of DO on N₂O emission, and relative quantities of autotrophic nitrifying denitrifying organisms and heterotrophic nitrifiers were suggested to have significance in affecting behaviors of activated sludge toward N₂O emission. On the other hand, serious accumulation of NO₂-N and NO₃-N resulted in high N₂O emission, to reduce NO₂-N and NO₃-N quickly and thoroughly was thus suggested for both reducing N₂O emission and improving nitrogen removal.

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The fast analysis of microcystin in environmental water was examined by using a protein phosphatase 2A inhibition assay. The heat treatment method was developed for rapid extraction of microcystin from Microcystis cells.
Quantitative analysis of microcystin by the protein phosphatase 2A inhibition assay required two hours. The assay was accurate and reproducible. The cultured Microcystis suspension was submitted to the protein phosphatase 2A inhibition assay as well as commercial ELISA assay kit with similar results. But sensitivity of this assay was not so good as the ELISA assay kit.
The microcystins were extracted from Microcystis cells by heat conditions such as temperature of 50-60 C° and heating time of more than 20 minutes. The heat treatment with α-amylase, cellulase and lysozyme was slightly facilitated to extraction of it.
Uses of the protein phosphatase 2A inhibition assay with the heat treatment method facilitate investigation of microcystin in natural water.

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The effects of electrochemical treatment on Microcystis extinction were studied. The test water flowed within a space between electrodes separated in parallel. Two kinds of test water were used : deionized water and sterilized water of Lake Kasumigaura. Cultured Microcystis cells were suspended in the two kinds of water, and the growth rate and the extinction rate of the cells were evaluated after the treatment. The extinction rate of the cells suspended in the lake water was much higher than that of the cells suspended in the deionized water. With the lake water, the extinction rate reached 98% by applying 10 V between the electrodes, and within 5minuites of the treatment time. Although it was not detected before the treatment, more than 10mg·l^-1 of free chlorine was detected from the lake water after the treatment. This implies that the free chlorine formed by the electrolysis of the water plays a key role. A good positive correlation between the extinction rate and the product of current density and the treatment time, seems to support the hypothesis.

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The water quality in marshes and streams around the coastal peatland at the Pacific seaboard of the Tokachi district, Hokkaido, Japan, has been examined. Water samples collected from the river affected by peatland were turning brown and showed specific characteristics proper to the peatland river water. The dissolved organic matter contents were significantly higher than those in the river less affected by peatland. However, the influence was relatively lower in the present peatland than the water quality in the peatland with wide area. The high ion exchange ability of peat also affected the composition of dissolving inos. Although water quality of the lake was similar to that of the river, the influence of saltwater was remarkable in the lake near seaboard. Significant positive correlation was found between dissolved organic matter contents and concentration of dissolved iron. However, it could not be concluded that only humic acid contributes to the chelating reaction with iron. It was inferred that organic anions play an important role on maintaining the balance of charges in the water samples affected by peatlands. It has been revealed that further study is needed on the quantification of non-humic substances.

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