Methane production by Methanothrix thermoacetophila via direct interspecies electron transfer with Geobacter metallireducens

Abstract

Methanothrix is widely distributed in natural and artificial anoxic environments and plays a major role in global methane emissions. It is one of only two genera that can form methane from acetate dismutation and through participation in direct interspecies electron transfer (DIET) with exoelectrogens. Although Methanothrix is a significant member of many methanogenic communities, little is known about its physiology. In this study, transcriptomics helped to identify potential routes of electron transfer during DIET between Geobacter metallireducens and Methanothrix thermoacetophila. Additions of magnetite to cultures significantly enhanced growth by acetoclastic methanogenesis and by DIET, while granular activated carbon (GAC) amendments impaired growth. Transcriptomics suggested that the OmaF-OmbF-OmcF porin complex and the octaheme outer membrane c-type cytochrome encoded by Gmet_0930, were important for electron transport across the outer membrane of G. metallireducens during DIET with Mx. thermoacetophila. Clear differences in the metabolism of Mx. thermoacetophila when grown via DIET or acetate dismutation were not apparent. However, genes coding for proteins involved in carbon fixation, the sheath fiber protein MspA, and a surface-associated quinoprotein, SqpA, were highly expressed in all conditions. Expression of gas vesicle genes was significantly lower in DIET- than acetate-grown cells, possibly to facilitate better contact between membrane-associated redox proteins during DIET. These studies reveal potential electron transfer mechanisms utilized by both Geobacter and Methanothrix during DIET and provide important insights into the physiology of Methanothrix in anoxic environments. IMPORTANCE Methanothrix is a significant methane producer in a variety of methanogenic environments including soils and sediments as well as anaerobic digesters. Its abundance in these anoxic environments has mostly been attributed to its high affinity for acetate and its ability to grow by acetoclastic methanogenesis. However, Methanothrix species can also generate methane by directly accepting electrons from exoelectrogenic bacteria through direct interspecies electron transfer (DIET). Methane production through DIET is likely to further increase their contribution to methane production in natural and artificial environments. Therefore, acquiring a better understanding of DIET with Methanothrix will help shed light on ways to (i) minimize microbial methane production in natural terrestrial environments and (ii) maximize biogas formation by anaerobic digesters treating waste.

Keywords: Geobacter; Methanothrix; acetate; archaea; cytochromes; direct interspecies electron transfer (DIET); granular activated carbon (GAC); magnetite; methane.

Fig 1

Growth of Geobacter metallireducens and Methanothrix thermoacetophila co-cultures with ethanol (20 mM) provided as the sole electron donor and CO₂ provided as the sole electron acceptor. Data represent means and standard deviations from triplicate cultures.

Fig 2

Morphology of Geobacter metallireducens and Methanothrix thermoacetophila co-culture aggregates. (A) Appearance of loose aggregates visible to the naked eye; (B and C) FISH images showing the close attachment of G. metallireducens (short rod, orange) cells to Mx. thermoacetophila (long filament, green); (D and E) Negative-stain TEM images of co-cultures; (F and G) Ultrathin TEM images of co-cultures. FISH, fluorescence in situ hybridization; TEM, transmission electron microscopy.

Fig 3

Effects of granular activated carbon (GAC) and magnetite on methane production by Methanothrix thermoacetophila. (A) Pure cultures using acetate (40 mM) as substrate in the presence of various GAC concentrations; (B) co-cultures using ethanol (20 mM) as substrate in the presence of GAC (40 g/L) or magnetite (10 mM); (C) pure cultures using acetate (40 mM) as substrate in the presence of magnetite (10 mM). Data represent the average and standard deviations from triplicate cultures.

Fig 4

Comparison of Geobacter metallireducens RNAseq libraries from co-cultures with Methanothrix thermoacetophila (MX), Methanosarcina barkeri (MB), Methanosarcina acetivorans (MA), Methanosarcina subterranea (MS), and Geobacter sulfurreducens (GS) using multidimensional scaling analysis with the biological coefficient of variation (BCV) method.

Fig 5

Proposed pathway for electron transfer in Geobacter metallireducens during DIET with Methanothrix thermoacetophila. Electrons are transferred from the quinone pool in the inner membrane to the CbcABCDE quinone-oxidoreductase complex, then to the periplasmic c-type cytochrome PpcA, which then shuttles electrons to the PccF (OmaF, OmbF, and OmcF) porin-cytochrome complex and then to the outer surface octaheme cytochrome encoded by Gmet_0930. Electrons may then be transferred directly to Mx. thermoacetophila. Arrows represent fold upregulated in DIET grown cells compared to cells grown with ethanol as the electron donor and ferric citrate as the electron acceptor (for proteins composed of multiple subunits, values from the most highly expressed subunits are shown). If an arrow is not listed with a protein from the proposed pathway, the gene was not differentially expressed between DIET- and Fe(III)-respiring cells. DIET, direct interspecies electron transfer.

Fig 6

Proposed pathways used by Methanothrix thermoacetophila during growth by acetoclastic and DIET-based methanogenesis in the presence or absence of magnetite. Arrows represent fold increase from median RPKM values (P values < 0.05) under DIET (red), acetate (blue), DIET+magnetite (green), and acetate+magnetite (orange) conditions. If proteins are composed of multiple subunits, values from the most highly expressed subunit are represented. Details regarding the fold differences and P values of each gene are provided in Table S3. CHO-MFR: formylmethanofuran; CHO-H₄MPT: formyltetrahydromethanopterin; CH≡H₄MPT: methenyltetrahydromethanopterin; CH₂=H₄MPT: methylenetetrahydromethanopterin; CH₃-H₄MPT: methyltetrahydromethanopterin; CH₃-S-CoM: 2-(methylthio)ethanesulfonate; Acetyl-CoA: acetyl-coenzyme A; 2-PGA: 2-phosphoglycerate; 3-PGA: 3-phosphoglycerate; BPG: 1,3-diphosphoglycerate; GAP: glyceraldehyde-3-phosphate; DHAP: dihydroxyacetone phosphate; FBP: fructose 1,6-bisphosphate; F6P: fructose 6-phosphate; Hu6P: D-arabino-3-hexulose-6-phosphate; Ru5P: ribulose-5-phosphate; HCHO: formaldehyde; RuBP: ribulose-1,5-bisphosphate; MP/MPH₂: oxidized/reduced forms of methanophenazine. DIET, direct interspecies electron transfer.