Micro-aeration unlocks endogenous nitrogen removal in anaerobic digesters via metabolically versatile fermentative bacteria.

Ammonia inhibition represents a significant bottleneck in the anaerobic digestion (AD) of high-nitrogen organic wastes. This study employed a batch reactor system subjected to alternating carbon-sufficient and carbon-limited phases to investigate whether pulsed micro-aeration, with a peak oxidation-reduction potential ranging from -150 to -50 mV, could promote the conversion of total ammonia nitrogen (TAN) to N2 in anaerobic digesters maintained under bulk anaerobic conditions, while simultaneously elucidating the competitive interplay between this nitrogen removal process and methanogenesis. The investigation integrated reactor performance monitoring, 15N stable-isotope tracing, and combined metagenomic and metaproteomic analyses. Compared to the strictly anaerobic control, the pulsed micro-aeration group exhibited a significant decrease in TAN concentration, with a maximum removal rate of 23.41 mg·L-1·d-1. Isotope analysis revealed that approximately 82.49% of the transformed 15NH4+-N was recovered as 15N2, thereby confirming the conversion of TAN to N2. The transient accumulation of NO2- and NH2OH, coupled with the absence of sustained NO3- accumulation and the enhanced expression of Hao, Nir, Nor, and Nos proteins, collectively suggested the involvement of a hydroxylamine/nitrite-associated pathway in conjunction with denitrification within the micro-aerated system. This nitrogen removal process was primarily mediated by native fermentative/facultative heterotrophic bacteria in the AD system, including Ottowia and Comamonas, which exhibited pronounced metabolic flexibility in response to carbon availability. When carbon was abundant, nitrogen removal was predominantly driven by heterotrophic denitrification; under carbon limitation, the system transitioned towards endogenous maintenance modes characterized by potential hydrogen utilization, mobilization of intracellular reserves (such as polyhydroxybutyrate and fatty acids), scavenging of residual organic matter, and the glyoxylate cycle. However, the activation of nitrogen removal through micro-aeration was accompanied by a suppression of methanogenesis, resulting in a methane loss of 1.31-2.77 mL per mg of TAN removed. Consequently, a paradigm of "mainstream methanogenesis-side-stream micro-aerobic nitrogen removal" is proposed, offering a novel strategy for developing robust processes to mitigate ammonia inhibition in AD.