Molecular characterizations of aquatic nitrogen-cycling bacterial communities
Sullivan, James Byron
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Investigations into the structure of aquatic bacterial communities involved in nitrogen cycling are necessary for the understanding and management of the systems in which they occur, as well as for their potential impact on global nitrogen dynamics. In aquatic systems, nitrogen moves from organism to organism via the dissolved nitrogen pool or by direct ingestion of biomass. At some point available nitrogen can be lost from a system. The loss of fixed nitrogen in aquatic systems occurs through physical removal or biogeochemical removal. It is the analysis of the bacterial communities responsible for the biogeochemical loss of fixed nitrogen that is the focus of this dissertation. In this study, DNA-based molecular methods are employed to investigate the occurrence and distribution of nitrogen cycling bacterial genes that code for enzymes involved in nitrification and denitrification. Initially, the ammonia-oxidizing bacterial community associated with finfish aquaculture in southern China is examined using T-RFLP and principal component analysis and correlations are drawn using various dissolved nitrogen species. These anaylses indicate that the ammonia oxidizing bacterial community is altered in water samples taken above the sediment below the fish cages. Next, the presence and phylogenetic distribution of nitrite reductase (nirK) gene sequences closely related to those recently found in marine ammonia-oxidizing isolates is examined in DNA extracted from an activated sludge wastewater treatment plant. Primers designed in this study from the ammonia oxidizer nirK sequences specifically amplify sequences that are very closely related to ones from which they were designed. These sequences are not closely related to known heterotrophic denitrifier nitrite reductase sequences and probably represent novel ammonia-oxidizer nirK genes. Finally, current prokaryotic in situ PCR methodology is improved upon and applied to the study of the microscale distribution of bacteria containing heterotrophic denitrification genes in activated sludge flocs. Nitrite reductase and nitrous oxide genes are amplified and detected inside intact bacterial cells. Additionally, an initial attempt at performing a simultaneous taxonomic and functional in situ identification is reported.