Tokyo Bay, a eutrophic bay in Japan, receives nutrients from wastewater plants and other urban diffuse sources via river input. A transect was conducted along a line from the Arakawa River into Tokyo Bay to investigate the ecological relationship between the river outflow and the distribution, abundance and population structure of ammonia-oxidizing bacteria (AOB). Five surficial marine sediments were collected and analysed with polyphasic approaches. Heterogeneity and genetic diversity of beta-AOB populations were examined using restriction fragment length polymorphism (RFLP) analysis of 16S rRNA and amoA genes. A shift of the microbial community was detected in samples along the transect. Both 16S rRNA and amoA genes generated polymorphisms in the restriction profiles that were distinguishable at each sampling site. Two 16S rRNA gene libraries were constructed using the reverse transcription polymerase chain reaction (RT-PCR) method to determine the major ammonia oxidizers maintaining high cellular rRNA content. Two major groups were observed in the Nitrosomonas lineage; no Nitrosospira were detected. The effort to isolate novel AOB was successful; the isolate dominated in the gene libraries. For quantitative analysis, a real-time PCR assay targeting the 16S rRNA gene was developed. The population sizes of beta-AOB ranged from 1.6 x 10(7) to 3.0 x 10(8) cells g(-1) in dry sediments, which corresponded to 0.1-1.1% of the total bacterial population. An immunofluorescence staining using anti-hydroxylamine oxidoreductase (HAO) antibody was also tested to obtain complementary data. The population sizes of ammonia oxidizers ranged between 2.4 x 10(8) and 1.2 x 10(9) cells g(-1) of dry sediments, which corresponded to 1.2-4.3% of the total bacterial fraction. Ammonia-oxidizing bacteria cell numbers deduced by the two methods were correlated (R = 0.79, P < 0.01). In both methods, the number of AOB increased with the distance from the river mouth; ammonia-oxidizing bacteria were most numerous at B30, where the ammonium concentration in the porewater was markedly lower and the nitrite concentration was slightly higher than nearby sites. These results reveal spatial distribution and shifts in the population structure of AOB corresponding to nutrients and organic inputs from the river run-off and phytoplankton bloom.

A polyphasic, culture-independent study was conducted to investigate the abundance and population structure of ammonia-oxidizing bacteria (AOB) in canal sediments receiving wastewater discharge. The abundance of AOB ranged from 0.2 to 1.9% and 1.6 to 5.7% of the total bacterial fraction by real-time PCR and immunofluorescence staining, respectively. Clone analysis and restriction endonuclease analysis revealed that the AOB communities influenced by the wastewater discharge were dominated by Nitrosomonas, were similar to each other, and were less diverse than the communities outside of the immediate discharge zone.Tokyo Bay is a representative enclosed eutrophic bay in the southern Kanto region of Japan. More than 86% of the shoreline is reclaimed, and artificial structures, such as canals and landfill islands, occupy the inner bay area (29). These artificial structures often hamper water exchange and accelerate precipitation of sludge containing excess organic matter, heavy metals, and toxic compounds (16,17,29). These human activities negatively affect the sea-bottom environment and limit natural remediation in the coastal ecosystem (19).Nitrification is essential to the nitrogen cycle in aquatic environments. When coupled with denitrification and/or anaerobic ammonium oxidation, it relieves the negative impacts of eutrophication through removal of nitrogen to the atmosphere as nitrous oxide or molecular nitrogen (5, 9, 38). However, nitrification is sensitive to environmental stress and contaminants (18, 23, 37). Ammonia-oxidizing bacteria (AOB) carry out the first, rate-limiting step of nitrification: conversion of ammonia to nitrite. The ecology and physiology of AOB are particularly difficult to study by conventional cultivation techniques because of their long generation times and low growth rates, which can result in underestimations of their numbers in the environment (21). Compared to the studies of AOB diversity in nature (1,4,6,12,13,22), quantitative studies have been limited, especially for marine environments (11). Therefore, a rapid, culture-independent detection technique for AOB would be useful for the study of marine AOB. Through this study, we developed and combined molecular and novel immunofluorescence staining approaches to investigate the spatial distribution, abundance, and population structure of AOB in a canal area of Tokyo Bay that is heavily polluted by excess organic and nutrient loading from wastewater treatment plant discharges. Particularly important for this study was quantification of AOB because of the deficiency of quantitative studies of AOB populations in marine environments

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Long-term trends of bottom-water concentrations of dissolved oxygen (DO), total nitrogen, and total phosphorus at 42 sites in Tokyo Bay were analyzed using monthly data from 1983 to 2018 measured by local government agencies. The results were visualized as spatial distribution maps. Bottom-water DO concentrations varied seasonally. The area of hypoxic bottom water (DO < 2 mg L −1 ) was greatest in September of every year. Examination of interannual variations of the spatial distribution of bottom-water DO concentrations in September revealed that along the eastern side of the bay, hypoxic zones have been shrinking since the mid-2000s, but along the western side of the bay, the bottom water closest to shore is hypoxic in September even today. The areal percentage of hypoxic bottom water during September within inner Tokyo Bay has declined from 30% around 2007 to < 15% around 2015 and has not changed since then. A comparison of the spatial distributions of the frequencies of occurrence of red tides (chlorophyll-a concentrations ≥ 50 μg L −1 ) and hypoxic water masses during 8-year intervals revealed that areas with high percentages of red tides coincided with areas with high percentages of hypoxic bottom water. The implication is that the reduction of the size of the hypoxic zones along the eastern side of the bay was due to less frequent occurrence of red tides there. Reducing the inputs of nutrients to the bay as a result of policies aimed at reducing water pollution has, therefore, contributed to the mitigation of the hypoxia problem.