Enrichment of ANME-2 Dominated Anaerobic Oxidation of Methane Coupled to Sulfate Reduction Consortia from Cold Seep Sediment (Ginsburg Mud Volcano, Gulf of Cadiz) in a Membrane Bioreactor

In this study, anaerobic methanotrophs (ANME) Archaea present in sediment from the Ginsburg mud volcano (Gulf of Cadiz), a known anaerobic oxidation of methane (AOM) site, was enriched in a membrane bioreactor (MBR). AOM activity coupled to sulfate reduction was monitored in the membrane bioreactor (MBR) for 726 days at 22 (± 3)°C. The MBR was equipped with a cylindrical ultrafiltration membrane, fed a defined medium containing artificial seawater and operated at a cross flow velocity of 0.02 m min⁻¹. Methane consumption was observed with simultaneous sulfate reduction at a volumetric rate of 0.7 mM of methane consumption d⁻¹ and 0.5 mM of sulfate reduction d⁻¹, respectively. The enriched biomass was tested for AOM activity during the end of the MBR operation. In batch incubations, when this biomass was incubated anaerobically with ¹³C labeled methane, ¹³C labeled inorganic carbon was produced and the AOM rate based on ¹³C-inorganic carbon production rate amounted to 1.2 μmol g_dw ⁻¹ d⁻¹. The incubation with ¹³C labeled carbonate showed an even higher rate of ¹³C-inorganic carbon production of 34.5 μmol g_dw ⁻¹ d⁻¹. Catalyzed reporter deposition - fluorescence in situ hybridization (CARD-FISH) analysis of the enriched biomass after ca. 400 days and 726 days, respectively, showed ANME-2 and Desulfosarcina type sulfate reducing bacteria were enriched in the MBR, which formed closely associated aggregates. The intermittent observation of acetate production in the bioreactor suggests that acetate produced during methane consumption might be an intermediate for electron transfer between the ANME and sulfate reducers.