Composition and Metabolic Potential of Fe(III)-Reducing Enrichment Cultures of Methanotrophic ANME-2a Archaea and Associated Bacteria

Abstract

The key microbial group involved in anaerobic methane oxidation is anaerobic methanotrophic archaea (ANME). From a terrestrial mud volcano, we enriched a microbial community containing ANME-2a, using methane as an electron donor, Fe(III) oxide (ferrihydrite) as an electron acceptor, and anthraquinone-2,6-disulfonate as an electron shuttle. Ferrihydrite reduction led to the formation of a black, highly magnetic precipitate. A significant relative abundance of ANME-2a in batch cultures was observed over five subsequent transfers. Phylogenetic analysis revealed that, in addition to ANME-2a, two bacterial taxa belonging to uncultured Desulfobulbaceae and Anaerolineaceae were constantly present in all enrichments. Metagenome-assembled genomes (MAGs) of ANME-2a contained a complete set of genes for methanogenesis and numerous genes of multiheme c-type cytochromes (MHC), indicating the capability of methanotrophs to transfer electrons to metal oxides or to a bacterial partner. One of the ANME MAGs encoded respiratory arsenate reductase (Arr), suggesting the potential for a direct coupling of methane oxidation with As(V) reduction in the single microorganism. The same MAG also encoded uptake [NiFe] hydrogenase, which is uncommon for ANME-2. The MAG of uncultured Desulfobulbaceae contained genes of dissimilatory sulfate reduction, a Wood–Ljungdahl pathway for autotrophic CO2 fixation, hydrogenases, and 43 MHC. We hypothesize that uncultured Desulfobulbaceae is a bacterial partner of ANME-2a, which mediates extracellular electron transfer to Fe(III) oxide.

Phenotypic and genomic characterization of the first alkaliphilic aceticlastic methanogens and proposal of a novel genus Methanocrinis gen.nov. within the family Methanotrichaceae

“…Many TMVs in this area have pH 8.5–9.1 and contain high concentrations of HCO and Na + ions (Lavrushin et al, 2021 ). The presence of Methanotrichaceae species had not previously been detected by 16S rRNA gene-based observations in several TMVs of this region (Merkel et al, 2021 ; Khomyakova et al, 2022 ; Slobodkin et al, 2023 ). However, in the other study, Methanothrix (“Methanosaeta”) thermophila accounted for almost 40% of Archaea in one TMV of southwestern Taiwan, suggesting the importance of this microbial group in certain TMVs (Cheng et al, 2012 ).…”

“…For the satellite bacteria, Whole Genome Shotgun projects have been deposited at DDBJ/ENA/GenBank under the BioProject ID PRJNA1010173. Metagenomic bioinformatics approaches were used to separate genomes in binary cultures as described in Slobodkin et al ( 2023 ). Metawrap v1.3 (Uritskiy et al, 2018 ) quant bins workflow was used to determine the relative abundance of the microorganisms in binary cultures by analyzing coverage using Salmon (Patro et al, 2017 ).…”

“…s rRNA gene and genome sequencing and analysis DNA from mud samples as well as from enrichment cultures was isolated using FastDNA Spin Kit for Soil according to the manufacturer's protocol (MP Biomedicals, Santa Ana, California, USA). Whole-genome sequencing and metagenomic bioinformatics analysis were used to assess the purity of cultures at the final stages of their purification according to methods described in Slobodkin et al (2023). 16S rRNA gene sequencing was performed as described previously (Slobodkina et al, 2022).…”

Convergence and horizontal gene transfer drive the evolution of anaerobic methanotrophy in archaea

“…Most descriptions of Methanovorans (e.g. ( 37 , 25 , 50 , 51 )) have relied 16S rRNA gene sequences to assign taxonomy, and thus MAGs which lack the 16S rRNA gene (like those from Dong et al ( 26 )) generally appear to be a methanogenic member of the Methanosarcinaceae . The introduction of the GTDB ( 52 ) has improved identification, with Methanovorans currently represented there by the g__DQIP01 annotation.…”

“…However, our observation of their potentially rapid and recent diversification at new seep sites like the Haakon Mosby Mud Volcano suggests that Methanovorans could be a useful case study through which to examine the processes shaping dispersal and diversity of methane seep taxa. Future analysis of a greater diversity of Methanovorans MAGs from new sampling sites such as the Glendhu Ridge MAG mentioned above and recently published MAGs from a terrestrial mud volcano ( 50 , 51 ) may clarify how such taxa spread and adapt to each new ecosystem they encounter.…”