Attempts to Identify Planctomycete Ammonium Oxidizing Bacteria in a Sequence Batch Reactor Sample

Abstract

Modern biological nitrogen removal processes focus on the use of anaerobic ammonium oxidation (anammox), where ammonium is converted to nitrogen gas under anoxic conditions. The discovery of anammox bacteria in many different environments has led to applications such as the single-reactor high-activity ammonia oxidation over nitrite (SHANON) and completely autotrophic nitrogen-removal over nitrate (CANON) reactors. Molecular approaches based on 16S rRNA, such as PCR and FISH, have overcome the limitations presented due to the slow growth rate of anammox organisms. This experiment aims to detect and identify any potential planctomycete anammox organisms that may be present in biofilm obtained from a sequence batch reactor, previously shown to contain various ammonium and nitrate oxidizing bacteria. DNA extracted from the biofilm was amplified using primers specific for the 16S rRNA of anammox planctomycetes. The pAA064 clone was created from the 1500bp amplified fragment, and the clone was digested with restriction enzymes and sequenced to identify the insert. Restriction enzyme digests indicated that the sequence did not belong to a known planctomycete anammox organism. The BLAST results showed that the sequence likely belongs to a spirochete or to a planctomycete anammox organism that is yet to be sequenced. One reason for the inconclusive identity of this organism is the large number of sequences from uncultured organisms or unidentified sequences from environmental samples. However, screening multiple clones to cover a wider range of organisms that may be present in the sample may overcome limitations in sequence databases.