Nicotinic acid enhances heavy metal stress resilience of wastewater anammox communities via NAD+ replenishment.

Anammox bacteria are vulnerable to heavy metals that are prevalent in nitrogen-rich industrial effluents, such as electroplating wastewater and landfill leachate, necessitating the development of cost-effective strategies to enhance stress resilience. Non-targeted metabolomics identified six candidate resilience-enhancing metabolites that were significantly up-regulated during and after Ni2+ inhibition. In lab-scale anammox reactors subjected to long-term Ni2+ stress, nicotinic acid (NA) exhibited the strongest resilience-enhancing effects, limiting the decline in anammox activity under prolonged Ni2+ stress to <20%, sustaining nitrogen removal efficiency above 60%, and shortening the recovery phase to <10 days. NA supplementation markedly increased the cellular NAD⁺ and ATP levels in anammox consortia by 7.6- and 2.1-fold, respectively, and genes involved in heavy metal efflux and anammox process were up-regulated, leading to community-level resilience enhancement. NA could also enhance the resilience of nitrifying bacteria in partial nitrification-anammox sludge under Ni2+ stress, with higher nitrification activity. Most anammox bacteria lack a complete de novo NAD⁺ biosynthesis pathway but possess the Preiss-Handler pathway that enables efficient conversion of NA into NAD⁺, leading to their strong responsiveness to exogenous NA. For wastewater microorganisms with similar NAD⁺ biosynthetic traits, NA supplementation represents a promising strategy to restore NAD⁺ pools, thereby improving resilience to heavy metal stress.