Conductive Particles Enable Syntrophic Acetate Oxidation between Geobacter and Methanosarcina from Coastal Sediments

Abstract

Coastal sediments are rich in conductive particles, possibly affecting microbial processes for which acetate is a central intermediate. In the methanogenic zone, acetate is consumed by methanogens and/or syntrophic acetate-oxidizing (SAO) consortia. SAO consortia live under extreme thermodynamic pressure, and their survival depends on successful partnership. Here, we demonstrate that conductive particles enable the partnership between SAO bacteria (i.e., Geobacter spp.) and methanogens (Methanosarcina spp.) from the coastal sediments of the Bothnian Bay of the Baltic Sea. Baltic methanogenic sediments were rich in conductive minerals, had an apparent isotopic fractionation characteristic of CO₂-reductive methanogenesis, and were inhabited by Geobacter and Methanosarcina As long as conductive particles were delivered, Geobacter and Methanosarcina persisted, whereas exclusion of conductive particles led to the extinction of Geobacter Baltic Geobacter did not establish a direct electric contact with Methanosarcina, necessitating conductive particles as electrical conduits. Within SAO consortia, Geobacter was an efficient [¹³C]acetate utilizer, accounting for 82% of the assimilation and 27% of the breakdown of acetate. Geobacter benefits from the association with the methanogen, because in the absence of an electron acceptor it can use Methanosarcina as a terminal electron sink. Consequently, inhibition of methanogenesis constrained the SAO activity of Geobacter as well. A potential benefit for Methanosarcina partnering with Geobacter is that together they competitively exclude acetoclastic methanogens like Methanothrix from an environment rich in conductive particles. Conductive particle-mediated SAO could explain the abundance of acetate oxidizers like Geobacter in the methanogenic zone of sediments where no electron acceptors other than CO₂ are available.IMPORTANCE Acetate-oxidizing bacteria are known to thrive in mutualistic consortia in which H₂ or formate is shuttled to a methane-producing Archaea partner. Here, we discovered that such bacteria could instead transfer electrons via conductive minerals. Mineral SAO (syntrophic acetate oxidation) could be a vital pathway for CO₂-reductive methanogenesis in the environment, especially in sediments rich in conductive minerals. Mineral-facilitated SAO is therefore of potential importance for both iron and methane cycles in sediments and soils. Additionally, our observations imply that agricultural runoff or amendments with conductive chars could trigger a significant increase in methane emissions.

Keywords: Desulfuromonadales; Geobacter; Methanosarcina; activated carbon; competitive exclusion; direct interspecies electron transfer; nanoSIMS; syntrophic acetate oxidation.

FIG 1

CO₂-reductive methanogenesis in the Bothnian Bay methanogenic zone. (a) The sampling site, RA2, was located off the Bothnian Bay northern coast. (b and c) Here, methane accumulated close to and sometimes over the saturation limit (b) and was strongly depleted in ¹³C (low δ¹³CH₄), which indicated a high apparent fractionation (α_C) characteristic of CO₂-reductive methanogenesis (c). Previous studies showed an α_C of ca. 1.05 (blue line) in Methanosarcina grown via CO₂-reductive methanogenesis (85, 86). An α_C of ca. 1.02 (orange line) was observed in Methanosarcina species grown by acetoclastic methanogenesis (87).

FIG 2

Incubation mixtures with and without activated carbon and representative organisms. (a) Quantitative PCR in original sediment samples showed that Desulfuromonadales were the dominant electrogens in the original sediment and in sediment slurries with conductive particles, but this group was almost extinct in a first slurry transfer without conductive particles. The only methanogens detected by qPCR in the original sediments were DIET-associated Methanosarcina, which remained abundant in slurry incubation mixtures with or without conductive particles. (b) In mud-free incubation mixtures with conductive GAC (sixth consecutive mud-free transfer), acetate was completely depleted after 63 days, and it was converted to methane with a high stoichiometric recovery (82%). Methanosarcina was the only Archaea genus detected in these mud-free cultures. Together, Methanosarcina and Geobacter represented ca. half of the microbial community, as determined by CARD-FISH. (c) On the other hand, in control incubation mixtures without conductive materials (third consecutive mud-free transfer), acetate consumption was much slower. Acetate was depleted after 150 days and converted to methane, with only 40% stoichiometric recovery. In control incubation mixtures without conductive GAC, Geobacter and Methanosarcina were led to extinction (Fig. S5F). Instead Methanothrix-like filamentous Archaea carried acetate utilization in control incubation mixtures without GAC (Fig. S5F).

FIG 3

Maximum likelihood trees of Bacteria and Archaea enriched in a seventh mud-free transfer with acetate and GAC. (a) A maximum likelihood tree of representative bacterial sequences from a mud-free transfer with conductive particles (GAC), under conditions strictly promoting methanogenic respiration. Acetate-oxidizing Desulfuromonadales dominated the 16S rRNA clone library, with more than half displaying close relationships to Geobacter psychrophilus (97% identity) and the rest to Desulfuromonas michiganensis (98%). The only methanogens enriched on acetate and GAC were relatives of Methanosarcina subterranea (99% identity), as shown in the maximum likelihood tree in panel b.

FIG 4

Experimental approach and evidence for SAO. (a) Experimental approach to distinguish between SAO and acetoclastic methanogenesis based on isotopic labeling. ¹³CH₃¹²COOH was provided as 10% of the total acetate, which played the role of the electron donor for SAO consortia from the Bothnian Bay. During SAO, acetate-oxidizing Geobacter cells are expected to produce ¹³CO₂ (¹³C, depicted in orange) and to incorporate [¹³C]acetate. During SAO, ¹³CO₂ will be diluted by the bicarbonate in the medium and should not generate significant ¹³CH₄. However, acetoclastic methanogenesis by Methanosarcina cells will generate ¹³CH₄ from ¹³CH₃¹²COOH, while cells incorporate [¹³C]acetate in their cell mass. Cells expected to incorporate [¹³C]acetate are encircled in orange. (b) SAO activity was validated by using labeled ¹³CO₂ production from acetate, especially in SAO consortia provided with GAC (blue) versus cultures without GAC (orange). (c) An overview of acetate catabolism and how much is used for respiration by Geobacter versus acetoclastic methanogenesis by Methanosarcina.

FIG 5

nanoSIMS identification of cells incorporating ¹³C-labeled acetate. (a and b) Highly abundant Geobacter cells (a) incorporated more ¹³CH₃¹²COOH per cell than Methanosarcina (b). Insets for panels a and b show percent assimilation in Geobacter (blue insets) and Methanosarcina (orange) over time. (c) Time-dependent distribution of cells labeled by Geobacter-specific probes compared with time-dependent incorporation of ¹³CH₃COOH in Geobacter cells (see scales below images) and an overlay of ¹³C incorporation (red) to total biomass as detected by tracing ³²S (green), using nanoSIMS. (d) Time-dependent distribution of cells labeled by Methanosarcina-specific probes compared with time-dependent incorporation of ¹³CH₃COOH in Methanosarcina-cells (see scales below images) and an overlay of ¹³C incorporation (red) to total biomass as detected by tracing ³²S (green) using nanoSIMS.

FIG 6

Syntrophic acetate-oxidizing bacteria cannot grow alone on acetate and GAC; they require the methanogen. If conductive GAC were sufficient for SAO bacteria to carry out acetate oxidation, the methanogenic inhibitor bromoethane sulfonate (BES) would collapse the rates of both methanogenesis (a) and acetate oxidation (b), indicating that the two processes are coupled and that Geobacter cannot grow alone on acetate and GAC. Methane production (a) and acetate utilization (b) rates were measured in cultures spiked with BES, in contrast to controls lacking BES and (c) a simplified representation of the BES inhibition effect on methanogenesis.

FIG 7

Model interactions with different treatments of a Baltic methanogenic community. Geobacter (green) and Methanosarcina (red) consortia competitively displaced Methanothrix-like (green) cells in Baltic sediments rich in iron-oxide minerals and in conductive particle-amended incubation mixtures. Geobacter was only present in incubation mixtures with conductive particles (Fig. S5F).