In Search of Anaerobic Ammonia Oxidation by Planctomycete-like Bacteria in Sequencing Batch Reactor Stressed by the Addition of Free Ammonia

Abstract

Planctomycetes belong to an unique anaerobic ammonia oxidation (anammox) member of the bacteria. They have a special organelle, anammoxosome, for anaerobic ammonia oxidation. Since they grow extremely slowly, there have been some difficulties identifying them. It is only recently that people have discovered planctomycetes might play a role in the process of converting ammonia and nitrite directly into dinitrogen without the usual conversion from nitrite to nitrate and to nitrogen gas. In a sequencing batch bioreactor treated by the addition of free ammonia, the concentration of nitrite initially spiked, and then overtime the nitrite was converted to nitrate by microorganisms. However, during this time, the level of Nitrobacter and Nitrospira detected was very low so it was speculated that planctomycetes may actually aid in this nitrifying process. DNA was first extracted from the cells, and then the DNA was amplified via PCR. The amplified DNA was cloned and colony hybridization was performed using probes Pla 46 and Amx 820. Fourteen clones hybridized with the probes. Five were sent for sequencing. None of these clones were planctomycetes due to incorrect hybridization temperature and more importantly, the washing temperature. This was likely that two of the clones were Actinobacteria, two were Proteobacteria (alpha and gamma) and one was Flavobacteria. The washing temperatures should have been increased (higher than the melting temperature of the probes) to remove sequences unspecifically hybridized to the target site. However, the result did suggest that the level of planctomycetes in the bioreactor was very low. Planctomycetes thus would not be responsible for the increase in nitrite reduction.