Microbially induced precipitation of silica by anaerobic methane-oxidizing consortia and implications for microbial fossil preservation

Abstract

Authigenic carbonate minerals can preserve biosignatures of microbial anaerobic oxidation of methane (AOM) in the rock record. It is not currently known whether the microorganisms that mediate sulfate-coupled AOM-often occurring as multicelled consortia of anaerobic methanotrophic archaea (ANME) and sulfate-reducing bacteria (SRB)-are preserved as microfossils. Electron microscopy of ANME-SRB consortia in methane seep sediments has shown that these microorganisms can be associated with silicate minerals such as clays [Chen et al., Sci. Rep. 4, 1-9 (2014)], but the biogenicity of these phases, their geochemical composition, and their potential preservation in the rock record is poorly constrained. Long-term laboratory AOM enrichment cultures in sediment-free artificial seawater [Yu et al., Appl. Environ. Microbiol. 88, e02109-21 (2022)] resulted in precipitation of amorphous silicate particles (~200 nm) within clusters of exopolymer-rich AOM consortia from media undersaturated with respect to silica, suggestive of a microbially mediated process. The use of techniques like correlative fluorescence in situ hybridization (FISH), scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM-EDS), and nanoscale secondary ion mass spectrometry (nanoSIMS) on AOM consortia from methane seep authigenic carbonates and sediments further revealed that they are enveloped in a silica-rich phase similar to the mineral phase on ANME-SRB consortia in enrichment cultures. Like in cyanobacteria [Moore et al., Geology 48, 862-866 (2020)], the Si-rich phases on ANME-SRB consortia identified here may enhance their preservation as microfossils. The morphology of these silica-rich precipitates, consistent with amorphous-type clay-like spheroids formed within organic assemblages, provides an additional mineralogical signature that may assist in the search for structural remnants of microbial consortia in rocks which formed in methane-rich environments from Earth and other planetary bodies.

Keywords: ANME-SRB; amorphous silica; methane seeps; microbial biomineralization; microfossils.

Fig. 1.

Epifluorescence and microscopy of sediment-free ANME-2/SRB consortia in laboratory incubations. (A–C) Correlated FISH, SEM, and EDS imaging of ANME-2/SRB consortia and associated Si-rich phases. Dashed ovals outline individual AOM consortia within the cluster across all three panels. (A) FISH image of a cluster of ANME-SRB consortia, with ANME-2 cells stained in pink and inferred SRB cells in blue. (B) Paired SEM image of the same cluster of AOM consortia in (A) where red arrows denote areas enriched in silica. Red Inset box: 2X magnified view of ~200 nm amorphous silica spheres. (C) EDS map showing elemental distribution, where yellow is carbon associated with ANME-SRB biomass, with silicon (blue) is concentrated in the extracellular matrix around individual AOM consortia. (D) Higher magnification SEM image showing nanoscale silica spheres associated with AOM consortia. (E and F) TEM cross-section of ANME-SRB consortia and closely associated Si-rich particles (red arrow).

Fig. 2.

Images of ANME-SRB consortia isolated from sediments. (A and B) NanoSIMS ¹²C¹⁴N maps; (C and D) NanoSIMS ²⁸Si maps of the same ANME-SRB consortia shown in (A and B), respectively, where silicon can be observed forming rims around the consortia. Lighter colors indicate higher counts, shown by the bars to the left of the images. (E) FISH of a sediment-associated ANME-SRB consortium, with ANME in pink and SRB in green. (F) SEM of the consortium in (E) after FIB section. (G) Elemental composition by EDS along a transect of the consortium in (F), which shows higher C and N relative abundances in the interior and an enrichment in Si and Al at the edges of the consortia.

Fig. 3.

Composition of Si-rich phases associated with ANME-SRB consortia isolated from sediments and in sediment-free incubations. (A) Elemental ratios of EDS-acquired compositional data of ANME-SRB consortium-attached Si-rich phases extracted directly from methane seep sediments (“ANME-SRB”) or grown under sediment-free conditions (“sed. free”) compared with the range of compositions of sediments from three different seep sediment locations from Northern and Southern California and the Costa Rican margin from which the AOM consortia were recovered (“sed.”). Sampling location details can be found in Materials and Methods. Reference clay mineral compositions are also noted [arrows on right; montmorillonite (mont), illite (ill), chlorite (chl)]. Also shown is the composition of three different Si-rich rings surrounding carbon-rich cell aggregates in a seep carbonate. (B) Predicted mineral stability diagram for silicate mineral formations in the experimental solution composition with both illite, smectite, and kaolinite included as clay minerals. Feldspar refers to albite; Chlorite(1) and Chlorite(2) refer to chamosite and clinochlore, respectively; and the red bar corresponds to ICP-MS measurement of [Si] in artificial seawater media from the sediment-free AOM enrichments. Crystalline or amorphous silica precipitation would require higher dissolved silica and lower pH values. (C) A general model of AOM consortia growth showing the estimated final number of consortia (contours) within a cluster as a function of the initial consortia numbers within the enrichment, constrained by the range of reported values for ANME-SRB doubling times (dashed lines).

Fig. 4.

Correlated epifluorescence microscopy, SEM, and elemental analysis of endolithic microbial consortia and associated Si-rich phases in seep carbonate. (A) DAPI-stained putative endolithic ANME-SRB consortia embedded within the seep carbonate matrix. (B) Correlated SEM of the same microbial consortia shown in (A). (C and D) EDS analysis of the same consortia as in (A) and (B) showing carbon rich biomass (yellow, C) surrounded by Si-rich rings in blue (white arrows, D). Numbered points correspond to EDS point spectra (Dataset S1) taken to compare elemental composition of rings to amorphous silica spheres attached to ANME-SRB consortia shown in Fig. 3A.

Fig. 5.

Correlated epifluorescence microscopy and elemental analysis of ANME-2-SRB consortia and associated Si-rich phases in seep carbonate. (A, B, and C) FISH-stained endolithic ANME-2-SRB consortia (white arrows) embedded within the seep carbonate matrix. Cells hybridized with the ANME-2 [ANME-2-712 (52)] and SRB [DSS658 (1)] specific FISH probes are stained in red and green, respectively. (D, E, and F) Carbon maps from EDS analysis of the consortia shown in (A, B, and C), respectively. (G, H, and I) Silica maps from EDS analysis of the consortia shown in (A, B, and C), respectively. White arrows point at Si-rims surrounding the consortia. Numbered points correspond to EDS point spectra (Dataset S1) taken to compare elemental composition of rings to amorphous silica spheres attached to ANME-SRB consortia shown in Fig. 3A.