Effects of magnetite on nitrate-dependent anaerobic oxidation of methane in an anaerobic membrane biofilm reactor: Metatranscriptomic analysis and mechanism prediction

Magnetite has been widely reported to accelerate methane production in anaerobic digestion. However, the effects of magnetite on the process of methane consumption, specifically nitrate-dependent anaerobic oxidation of methane (AOM), and its under-lying mechanisms remain poorly understood. In this study, the addition of magnetite significantly improved the removal of nitrate in an anaerobic membrane biofilm reactor (AnMBfR) (25.2 & PLUSMN; 2.5 mg NO-3-N/L/d) compared to that in an AnMBfR without magnetite (8.7 & PLUSMN; 4.2 mg NO-3-N/L/d). Metatranscriptomic analysis showed that genes encoding type IV pilus assembly proteins were highly expressed in nitrate/nitrite-reducing bac-teria in the biofilms of the two AnMBfRs. Specifically, the transcript abundance of these genes was observed to be higher in the presence of magnetite (gene expression levels, log2FPKM = 13.08) compared to the absence of magnetite (12.04). Biofilms with magnetite exhibited a linear increase in electron transfer resistance with increasing temperature, and the decrease in pH significantly increased their conductivity, indicating a metallic-like conductivity. Contrarily, an increase in temperature or decrease in pH did not influence the resistance or conductivity of biofilms without magnetite. Some of these cytochrome c (CYC)-like proteins were electrically active in the electrochemical Fourier transform infrared spectra and were closely associated with the outer-membrane multi-heme c-type cytochromes (OMCs). The intensities of the OMC-like bands in the surface-enhanced resonance Raman spectra with biofilms in the presence of magnetite were higher than those without magnetite. This study suggests that magnetite pro -motes the nitrate-dependent AOM process because of the formation of methanotrophic consortia based on direct interspecies electron transfer (DIET).& COPY; 2023 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).