Fe0/Fe2+-Dependent Nitrate Reduction in Anammox Consortia Questions the Enzymatic Mechanism of Nitrate Reduction by Anammox Bacteria

The anammox bacteria (AnAOB) have been suggested to perform dissimilatory Fe-0/Fe2+-dependent nitrate reduction to ammonium (DNRA), with nitrite as intermediate. However, evidence at genetic and transcriptional levels for how AnAOB reduces nitrate is still lacking, making their roles in this process obscure and questionable. The short-term batch assays and long-term operation of bioreactor combined with 16S rDNA sequencing and transcription analysis were used for the first time to investigate the nitrate reduction pathway in anammox consortia. It is shown that AnAOB metabolically well grew with nitrate as the sole nitrogen source. They reduced nitrate to nitrite with highly expressed narG gene. However, the DNRA-enabling gene nrfA was not expressed by AnAOB. Abiotic reduction by Fe-0/Fe2+ contributed to the nitrate and nitrite reduction to ammonium. The correlation analysis of microbial community structure and metabolic functions indicated that amino acid and carbohydrate transport and metabolism interactions in coexisting bacteria could facilitate the AnAOB growth with nitrate as the only nitrogen source. Our findings suggest that the contribution of enzymatic DNRA by AnAOB to the observed nitrate reduction has been probably overestimated in previous publications, and it should be questioned until the clear enzymatic mechanism responsible for nitrate reduction is identified.