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 toxic heavy metal frequently detected in wastewater, often alongside nitrate (NO₃ ^-). Nitrate-dependent anaerobic methane oxidation (N-DAMO) is a promising process for the simultaneous removal of methane (CH₄) and NO₃ ^- in wastewater treatment plants. Because Cr-(VI) can serve as an alternative electron acceptor, its presence may alter the N-DAMO performance. Here, we investigated the impact of Cr-(VI) on an enrichment culture containing <i>Candidatus Methanoperedens</i> and <i>Candidatus Methylomirabilis</i>, using NO₃ ^- as the electron acceptor and ¹³C-CH₄ as the electron donor. Cultures were exposed to varying Cr-(VI) concentrations, and microbial activity was assessed using GC-MS, 16S rRNA gene sequencing, and qPCR. Cr-(VI) was reduced within the cultures, but this reduction was not linked to CH₄ oxidation. Instead, CH₄ oxidation was significantly inhibited, with declines in the relative abundances of both N-DAMO organisms. Cr-(VI) reduction was likely mediated by denitrifiers through nitrate reductase activity or abiotically via the reaction with nitrite (NO₂ ^-). These findings reveal functional resilience of microbial consortia in contaminated environments but highlight Cr-(VI) toxicity as a constraint for N-DAMO-based wastewater treatment.