Characterization and quantification of ammonia-oxidizing bacteria in eutrophic coastal marine sediments using polyphasic molecular approaches and immunofluorescence staining

Abstract

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.