Anaerobic ammonium oxidation coupled to iron­(III) reduction, termed Feammox, is a newly discovered nitrogen cycling process. However, little is known about the roles of electron shuttles in the Feammox reactions. In this study, two forms of Fe­(III) (oxyhydr)­oxide ferrihydrite (ex situ ferrihydrite and in situ ferrihydrite) were used in dissimilatory Fe­(III) reduction (DIR) enrichments from paddy soil. Evidence for Feammox in DIR enrichments was demonstrated using the 15N-isotope tracing technique. The extent and rate of both the 30N2–29N2 and Fe­(II) formation were enhanced when amended with electron shuttles (either 9,10-anthraquinone-2,6-disulfonate (AQDS) or biochar) and further simulated when these two shuttling compounds were combined. Although the Feammox-associated Fe­(III) reduction accounted for only a minor proportion of total Fe­(II) formation compared to DIR, it was estimated that the potentially Feammox-mediated N loss (0.13–0.48 mg N L–1 day–1) was increased by 17–340% in the enrichments by the addition of electron shuttles. The addition of electron shuttles led to an increase in the abundance of unclassified Pelobacteraceae, Desulfovibrio, and denitrifiers but a decrease in Geobacter. Overall, we demonstrated a stimulatory effect of electron shuttles on Feammox that led to higher N loss, suggesting that electron shuttles might play a crucial role in Feammox-mediated N loss from soils.

Microorganisms are major drivers of elemental cycling in the biosphere. Determining the abundance of microbial functional traits involved in the transformation of nutrients, including carbon (C), nitrogen (N), phosphorus (P) and sulfur (S), is critical for assessing microbial functionality in elemental cycling. We developed a high-throughput quantitative-PCR-based chip, Quantitative microbial element cycling (QMEC), for assessing and quantifying the genetic potential of microbiota to mineralize soil organic matter and to release C, N, P and S. QMEC contains 72 primer pairs targeting 64 microbial functional genes for C, N, P, S and methane metabolism. These primer pairs were characterized by high coverage (average of 18-20 phyla covered per gene) and sufficient specificity (>70% match rate) with a relatively low detection limit (7-102 copies per run). QMEC was successfully applied to soil and sediment samples, identifying significantly different structures, abundances and diversities of the functional genes (P<0.05). QMEC was also able to determine absolute gene abundance. QMEC enabled the simultaneous qualitative and quantitative determination of 72 genes from 72 samples in one run, which is promising for comprehensively investigating microbially mediated ecological processes and biogeochemical cycles in various environmental contexts including those of the current global change.