Hexavalent Chromium Inhibits Nitrate-Dependent Anaerobic Methane Oxidation While Enriching Denitrifiers: Insights into Microbial Interactions for Simultaneous Methane, Nitrate and Chromate Removal

Chromate (Cr(VI)) is a widely distributed heavy metal known for its toxicity and adverse effects on human health. Due to its extensive industrial use, Cr(VI) is commonly detected in municipal and industrial wastewater, often co-occurring with other contaminants such as nitrate (NO ₃ ⁻ ). Nitrate-dependent anaerobic methane oxidation (N-DAMO) is a promising microbially driven process that enables the simultaneous removal of methane (CH ₄ ) and NO ₃ ⁻ , potentially lowering the carbon footprint of wastewater treatment plants (WWTPs). Given that Cr(VI) can act as an alternative electron acceptor, its presence may influence the N-DAMO process either by supporting CH ₄ oxidation or, conversely, by exerting toxic effects that inhibit microbial activity. In this study, we investigated the impact of Cr(VI) on an N-DAMO enrichment culture composed of Candidatus Methanopereden s and Candidatus Methylomirabilis, using NO ₃ ⁻ as the electron acceptor and ¹³ C-CH4 as the sole electron donor. Cultures were exposed to varying concentrations of Cr(VI), and microbial activity was assessed through GC-MS, 16S rRNA gene sequencing and qPCR. Here we found that while Cr(VI) was reduced within the culture, this process was not coupled to CH ₄ oxidation. In fact, CH ₄ oxidation was significantly inhibited, and the relative abundances of Ca . Methanoperedens and Ca . Methylomirabilis declined under increasing Cr(VI) concentrations. Cr(VI) reduction was likely mediated by the flanking microbial community via two possible pathways: (i) enzymatically, through nitrate reductase activity in denitrifying bacteria, and (ii) abiotically, through chemical reaction with nitrite (NO ₂ ⁻ ), an intermediate of denitrification. These findings highlight the potential of N-DAMO microbes to confer functional resilience in contaminated environments. However, the sensitivity of methanotrophic organisms to Cr(VI) underscores a limitation in applying this process to wastewater treatment systems containing heavy metals.