Molecular investigation of Ammonia oxidizing bacteria in different environments based on the Ammonia monooxygenase (amoA) gene

Abstract

Aerobic chemolithoautotrophic Ammonia-Oxidizing Bacteria (AOB) are able to convert ammonia into nitrite during the nitrification process. The ecological relevance of these biological functional groups is due to the essential role they play in the global nitrogen cycle.<br />Ammonia-Oxidizing Bacteria communities were studied in two different environments in Austria, an engineered- and a natural system. Activated sludge samples were collected from the biological second stage during the start-up phase of a nitrification/denitrification coupled process, working at full operation scale in the main waste water treatment plant in Vienna. Soil and water samples were collected from the LKAS 2 catchment, an alpine mountainous system located in the so called Northern Calcareous Alps in Austria reaching altitudes between 1200 up to 2000 m. The microbial population diversity was analyzed by polymerase chain reaction, followed by denaturing-gradient gel electrophoresis (DGGE) and cloning, using the ammonia monooxygenase (amoA) gene as a functional molecular marker. Sequencing analyses revealed Nitrosomonas oligotropha cluster 6 as the dominant group in the WWTP and the bacterial community developed after seven weeks. The decrease of the ammonia nitrogen (NH4-N) concentration of the WWTP effluent was associated to the establishment of the Ammonia-Oxidizing Bacteria (AOB) indicating an active biological nitrification process. Molecular analysis of soil samples showed a highly diverse bacterial community with a few number of dominant species. Higher richness of Nitrosospira clusters 0, 2, 3 and 4 was confirmed by sequencing analysis of excised DGGE bands. Nitrosovibrio and Nitrosomonas oligotropha cluster 6 were also found in soil samples. Band patterns of spring water samples suggested the presence of members of the genus Nitrosospira and a faint band might indicate presence of members of the Nitrosomonas lineage.<br />The present work demonstrate the applicability of the amoA PCR/DGGE approach in monitoring the changes of complex bacterial AOB communities and represent a powerful tool for a better understanding of the richness, distribution and population dynamics of these important microorganisms in the environment.