Enrichment of nitrogen cycle bacteria from the biofilter of a freshwater recirculating aquaculture system. aquaculture biofilter anaerobic ammonium oxidizer enrichment

Study description: The accumulation of nitrogenous compounds is a major problem in many recirculating aquaculture systems (RAS) and in most cases biofiltration is used to remove the nitrogen from the water. In this study, nitrogen cycle (N-cycle) bacteria were enriched from a sample taken at the anaerobic compartment of a biofilter connected to a RAS. Filtered water from the aquaculture system, supplemented with ammonium, nitrite and nitrate (concentrations not exceeding 500 µM), was fed to the reactor. A stable enrichment culture was obtained which anaerobically converted ammonium, nitrite and nitrate into dinitrogen gas. Metagenome sequencing showed the abundant presence of 3 different types of N-cycle bacteria: a Brocadia species (anaerobic ammonium oxidizer), a Nitrospira species (nitrite oxidizer) and a Denitratisoma species (denitrifyer). Sequencing: Metagenomic sequencing of the reactor was performed on the IonTorrent Personal Genome Machine system (PGM, Life Technologies, Bleiswijk, the Netherlands) according to the manufacturer’s instructions (unless specified otherwise). To avoid biases caused by genomic DNA isolation, DNA obtained by the CTAB based extraction method (1 run) and by the Powersoil kit (2 runs) was sequenced. All three runs were performed on an Ion 318 chip using the 200bp protocol. 1 µg of genomic DNA extracted using the CTAB-based extraction method was sheared for 1 min using Ion shear chemistry, adapter ligated and size selected using an E-Gel SizeSelect Agarose gel (Life Technologies, Bleiswijk, the Netherlands). The size-selected library was amplified for 8 cycles prior to template preparation. Template preparation and sequencing were performed according to the manufacturer’s instructions. DNA (1 µg) obtained with the powersoil kit was sheared for 10 min and 15 min, respectively. All other steps were performed as described above.