Temperature-induced metabolic reconfiguration and community shifts reveal the mechanistic basis of cold inhibition on anammox consortia

Abstract

Temperature fluctuation is a major constraint on the stability of anammox systems, yet the cellular and community-level mechanisms underlying cold inhibition remain poorly understood. In this study, a 159-d continuous-flow anammox reactor was operated under sequential temperature shifts (35 °C \to 25 °C \to 15 °C) to elucidate the responses of microbial metabolism, enzyme conformation, and community interactions. Low temperature (15 °C) markedly suppressed nitrogen removal performance, with effluent NH₄⁺-N and NO₂⁻-N concentrations increasing threefold. Intracellular cofactors (NAD⁺/NADH, NADP⁺/NADPH) and heme c content declined sharply, while extracellular polymeric substances accumulated, indicating energy reallocation from metabolism to structure maintenance. Molecular dynamics simulations revealed that hydroxylamine oxidoreductase exhibited reduced conformational flexibility and distorted redox centers at 15 °C, explaining the enzymatic activity loss. Metagenomic and qPCR analyses showed that the abundance of hdh and other anammox functional genes decreased by over 80%, accompanied by a community shift from Candidatus Kuenenia to psychrophilic Pseudomonadota and denitrifying Denitratisoma. Functional pathway analysis highlighted the downregulation of oxidative phosphorylation and quorum sensing, and the upregulation of stress-response and transport systems. Collectively, these results reveal a multi-level adaptation strategy and identify the molecular basis of cold inhibition in anammox consortia, providing theoretical guidance for improving nitrogen removal resilience in low-temperature environments.

Graphical abstract

Keywords

Anammox / Temperature change / Energy metabolism / Community structure

Highlight

	● Low temperature suppressed anammox activity and depleted NAD+/NADPH and heme c.
	● HAO lost structure flexibility and redox stability at 15 °C.
	● Cold stress induced microbial shifts from Candidatus Kuenenia to Pseudomonadota .
	● Metagenomics revealed downregulated oxidative phosphorylation and quorum sensing.

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