High-Performance Solar-Driven Residual Nitrate Removal by a DNRA-Anammox Self-Loop in Anammox Bacteria toward Sustainable Nitrogen Removal from Wastewater.

Abstract

Residual nitrate remains a stoichiometric bottleneck limiting advanced nitrogen removal in anaerobic ammonium oxidation (anammox) processes. The nitrate-reducing and electroactive traits of anammox bacteria (AnAOB) offer a promising strategy to polish residual nitrate in situ using external electrons. However, whether AnAOB can harvest photoelectrons and overcome multilayered trans-envelope barriers in nanobiohybrids remains unresolved. Here, we engineered dual-site AnAOB-cadmium sulfide (CdS) interfaces on the outer membrane and anammoxosome membrane of high-purity planktonic AnAOB (>95%) to enable efficient nitrate reduction and removal. The AnAOB-CdS biohybrid achieved complete nitrate reduction and increased total nitrogen removal to 98.2 ± 0.9% without N2O emission, exceeding the stoichiometric limit of the anammox reaction (89%). Isotopic tracing and metatranscriptomics supported the activation of a dissimilatory nitrate reduction to ammonium (DNRA)-anammox self-loop, with upregulation of DNRA (napA, 4.95-fold; nrfA, 9.82-fold) and anammox (hdh, 1.79-fold; hzsABC, 2.59-fold) genes. Mechanistically, "Candidatus Brocadia sp." captured extracellular photoelectrons through outer membrane c-type cytochromes and self-secreted riboflavin to drive periplasmic nitrate reduction. This speculation is supported by the 1.77- and 1.95-fold upregulation of extracellular monoheme and pentaheme c-type cytochromes, respectively, while extracellular riboflavin accumulated to 150 nM. Reverse photoelectron transport elevated the NADH/NAD+ ratio by 3.50-fold; this pool of reducing equivalents, combined with endogenously generated photoelectrons, drives nitrite-to-ammonium reduction within the anammoxosome. This work provides a solar-driven biohybrid for high-efficiency residual nitrate removal and elucidates the previously unknown photoelectron uptake mechanisms in AnAOB, offering a sustainable strategy toward advanced nitrogen removal in wastewater treatment.