Significant progress in understanding the key enzymes or species of anammox has been made; however, the nitrogen removal mechanism in complex coupling systems centered on anammox remains limited. In this study, by the combination of metagenomics−metatranscriptomics analyses, the nitrogen removal in the anammox-centered coupling system that entails partial denitrification (PD) and hydrolytic acidification (HA, A-PDHA) was elucidated to be the nitrogen transformation driven by the electron generation−transport−consumption process. The results showed that a total nitrogen (TN) removal efficiency of >98%, with a TN effluence of <1 mg/L and a TN removal contribution via anammox of >98%, was achieved after 59 days under famine operation and alkaline conditions during the start-up process. Further investigation confirmed that famine operation promoted the activity of genes responsible for electron generation in anammox, and increased the abundance or expression of genes related to electron consumption. Alkaline conditions enhanced the electron generation for PD by upregulating the activity of glyceraldehyde 3-phosphate dehydrogenase and strengthened electron transfer by increasing the gene encoding quinone pool. Altogether, these variations in the electron flow led to efficient nitrogen removal. These results improve our understanding of the nitrogen removal mechanism and application of the anammox-centered coupling systems in treating nitrogen wastewater.

The anammox-based technologies are generally inhibited by refractory dissolved organic matter (rDOM), which is ubiquitous in real wastewater. In this study, a novel cost-effective approach, namely, zero-valent iron (ZVI) treatment, was presented to alleviate such inhibitory effects. The results showed that ZVI mitigated the inhibition of fulvic acids (FA) to anammox. Due to the 2 g/L ZVI addition, the nitrogen removal efficiency (NRE) increased from 83.53 to 90.06% when the FA concentration increased from 0 to 160 mg/L in wastewater. Additionally, the co-occurrence network linking Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) and 16S rRNA sequencing results revealed the mechanism of ZVI mitigating inhibition by promoting the reduction of nitro and nitroso groups of FA and reshaping the metabolic division of functional bacteria. Hydrolytic acidifying bacteria (e.g., Anaerolineaceae and Ignavibacterium) were enriched for FA degradation, which was ultimately beneficial for denitrifying bacteria and anaerobic ammonium oxidation bacteria (AnAOB). Furthermore, metagenomics revealed that ZVI stimulated multiple nitrogen removal formed by anammox, denitrification, and DNRA by accelerating extracellular electron transfer resorting to FA serving as an electron shuttle and upregulating functional genes encoding electron generation, transport, and consumption processes.

This study introduced photogenerated electrons into the anammox system by coupling them to a g-C3N4 nanoparticle photocatalyst. A high nitrogen removal efficiency (94.25%) was achieved, exceeding the biochemical limit of 89% imposed by anammox stoichiometry. Photogenerated electrons boosted anammox metabolic activity by empowering key enzymes (NIR, HZS, and WLP-related proteins) and triggered rapid algal enrichment by enhancing the algal Calvin cycle, thus developing multiple anammox–algae synergistic nitrogen removal processes. Remarkably, the homologous expression of cbb3-type cytochrome c oxidase (CcO) in anammox bacteria was discovered and reported in this study for the first time. This conferred aerobic respiration capability to anammox bacteria and rendered them the principal oxygen consumer under 7.9–19.8 mg/L dissolved oxygen, originating from algal photosynthesis. Additionally, photogenerated electrons selectively targeted the cb1 complex and cbb3-type CcO as activation sites while mobilizing the RegA/B regulatory system to activate the expression of cbb3-type CcO. Furthermore, cbb3-type CcO blocked oxidative stress in anammox by depleting intracellular oxygen, a substrate for reactive oxygen species synthesis. This optimized the environmental sensitivity of anammox bacteria and maintained their high metabolic activity. This study expands our understanding of the physiological aptitudes of anammox bacteria and provides valuable insights into applying solar energy for enhanced wastewater treatment.

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