Newly designed high-coverage degenerate primers for nitrogen removal mechanism analysis in a partial nitrification-anammox (PN/A) process

Reliable tools for quantification of different functional populations are required to achieve stable, effective nutrients removal in partial nitrification and anammox (PN/A) processes. Here we report the design and validation of degenerate PCR primer pairs targeting anammox bacteria, aerobic ammonium-oxidizing bacteria (AeAOB) and nitrite-oxidizing bacteria (NOB) with high coverage but without sacrificing specificity. The new primer pairs are able to cover a broader range of the targeted populations (58.4 vs 21.7%, 49.5 vs 47.6%, 80.7 vs 57.2% and 70.5 vs 42.3% of anammox bacteria, AeAOB, Nitrobacter and Nitrospina, respectively) than previously published primers. Particularly, the Amx719F/875R primer can retrieve a larger number of 16S rRNA genes from different types of samples with amplicons covering all known anammox bacteria genera (100% coverage) including the recently found novel genus, Asahi BRW. These newly desinged primers will provide a more reliable molecular tool to investigate the mechanisms of nitrogen removal in PN/A processes, which can provide clearer links between reactor performance, the metabolic activities and abundances of functional populations, shedding light on conditions that are favorable to the establishment of stable PN/A.