Biogeochemistry and community composition of iron- and sulfur-precipitating microbial mats at the Chefren mud volcano (Nile Deep Sea Fan, Eastern Mediterranean)

Abstract

In this study we determined the composition and biogeochemistry of novel, brightly colored, white and orange microbial mats at the surface of a brine seep at the outer rim of the Chefren mud volcano. These mats were interspersed with one another, but their underlying sediment biogeochemistries differed considerably. Microscopy revealed that the white mats were granules composed of elemental S filaments, similar to those produced by the sulfide-oxidizing epsilonproteobacterium "Candidatus Arcobacter sulfidicus." Fluorescence in situ hybridization indicated that microorganisms targeted by a "Ca. Arcobacter sulfidicus"-specific oligonucleotide probe constituted up to 24% of the total the cells within these mats. Several 16S rRNA gene sequences from organisms closely related to "Ca. Arcobacter sulfidicus" were identified. In contrast, the orange mat consisted mostly of bright orange flakes composed of empty Fe(III) (hydr)oxide-coated microbial sheaths, similar to those produced by the neutrophilic Fe(II)-oxidizing betaproteobacterium Leptothrix ochracea. None of the 16S rRNA gene sequences obtained from these samples were closely related to sequences of known neutrophilic aerobic Fe(II)-oxidizing bacteria. The sediments below both types of mats showed relatively high sulfate reduction rates (300 nmol x cm(-3) x day(-1)) partially fueled by the anaerobic oxidation of methane (10 to 20 nmol x cm(-3) x day(-1)). Free sulfide produced below the white mat was depleted by sulfide oxidation within the mat itself. Below the orange mat free Fe(II) reached the surface layer and was depleted in part by microbial Fe(II) oxidation. Both mats and the sediments underneath them hosted very diverse microbial communities and contained mineral precipitates, most likely due to differences in fluid flow patterns.

FIG. 1.

(A) Bathymetric map of the Nile Deep Sea Fan. The circle indicates the position of the Menes Caldera. (Adapted from reference with permission of Geosciences Azur/CIESM.) (B) Bathymetric map of the Menes Caldera with its three mud volcano systems. (C) Bathymetric map of the Chefren mud volcano. “X” indicates the location close to Chefren characterized by orange and white mats, and the circle indicates the location where brine samples were obtained during Nautile dive 18. The maps in panels B and C were obtained during METEOR expedition BIONIL M70/2 in 2006 using the EM120 multibeam.

FIG. 2.

Microbial mat system of the brine-impacted seep at the rim of the Chefren mud volcano. (A) Photograph taken by the submersible Nautile at the site of recovery of the orange and white mats. On the right side, the sediments are populated by sessile worms (arrows) forming tubes from sediment particles. Scale bar = 3 m. (B) Close-up of the mat system. Brine flowed downward (arrows) from the steep rim of the Chefren mud volcano across the white mats. Scale bar = 1 m. (C) Photographs of a core from the white mat. (Left) Black to gray sediment layers below the white mat. (Right) Top of the core. The white mat was composed of cotton ball-like precipitates overlying black fluidic sediments. Scale bar = 3 cm. (Inset) Small motile polychaetes associated with the white mat and the black sediment layer. The red color of the polychaetes indicates elevated hemoglobin levels, a typical adaptation to reduced sediments. Scale bar = 0.5 cm. (D) Photographs of a core from the orange mat. (Left) Greyish sediment layers below the orange mat. (Right) Top of the core. The arrows indicate a sessile worm (top arrows) and the orange fluffy material and flakes overlaying grayish sediments (bottom arrow). Scale bar = 3 cm. (E) Orange precipitates on sediments at the border of the brine lake. Scale bar = 20 cm. (F) Dense white mats floating on top of the large brine lake filling the center of the Chefren mud volcano. Scale bar = 3 m. (Panels A, B, E, and F were taken by the Nautile submersible during the 2003 Nautinil campaign [photos courtesy of Ifremer].)

FIG. 3.

(A) Dissecting microscope image of an S aggregate from the white mat. Scale bar = 1 mm. (B) Phase-contrast image of S filaments from the white mat. Scale bar = 10 μm. (C) High-resolution SEM image of filaments and associated cells from the white mat. (D) FISH image showing Arc94-targeted cells (green). (E) Image of Fe oxide flakes from the orange mat. Scale bar = 1 mm. (F) Light microscope image of an orange flake. (G) High-resolution SEM image of damaged sheaths from a flake. The arrows indicate two distinct types of sheathed bacteria (bacteria are not visible, but their sheaths are). (H) FISH image showing Mγ705-targeted sheaths. The arrows indicate two distinct types of sheathed bacteria similar to C. fusca (arrow 1) and C. polyspora (arrow 2). (B to D and F to H) Scale bars = 10 μm. Cores NL18PC3(5) and NL18PC1(8) were used for microscopy.

FIG. 4.

Cl⁻ profiles from fluid flow models. (A) Measured Cl⁻ profile from underneath the white mats (circles), modeled Cl⁻ profile at a constant flow of 15 m·a⁻¹ (straight line), and modeled Cl⁻ profile after 10 h with a fluid flow velocity of zero (dashed line). (B) Measured Cl⁻ profile from underneath the orange mats (circles), modeled Cl⁻ profile at a constant flow of 0.6 m·a⁻¹ (straight line), and modeled Cl⁻ profile after 10 h with a fluid flow velocity of zero (dashed line).

FIG. 5.

(A) Replicate SR rates (circles) and sulfate concentrations underneath the white mats. (B) Replicate rates of methane oxidation (circles) and methane concentrations underneath the white mats. (C) Fe(II) (open circles) and HS⁻ (filled circles) concentrations underneath the white mats. (D) Sedimentological description of the sediment underneath the white mats. (E) Replicate SR rates (circles) and sulfate concentrations underneath the orange mats. (F) Replicate methane oxidation rates (circles) and methane concentrations underneath the orange mats. (G) Fe(II) (open circles) and HS⁻ (filled circles) concentrations underneath the orange mats. (H) Sedimentological description of the sediment underneath the orange mats.

FIG. 6.

(A) Double hybridization using FISH probes ANME2-538 (red) and DSS658 (green). (B) ANME3-1249-targeted cells. (C) DSS658-targeted cells. (D) Mγ705-targeted single cells. Bars = 10 μm. Cores NL18PC3(5) and NL18PC1(8) were used for microscopy.

FIG. 7.

Maximum parsimony tree of 16S rRNA gene sequences from Deltaproteobacteria obtained in this study, as well as from the GenBank database. The names in brackets are those of well-known cold seeps and hydrothermal vents. The bootstrap values at the nodes are percentages based on 500 replicates. Sequences obtained in this study are indicated by bold type, and the numbers in parentheses indicate the numbers of sequences with 98% identity to the relevant sequences from the white mat, the sediment underlying the white mat, the orange mat, and the sediment underlying the orange mat. Only selected sequences are displayed. Sequences that are targeted by the DSS658 and 660 probes are indicated.

FIG. 8.

Maximum parsimony tree of 16S rRNA gene sequences from Gamma-, Beta-, and Epsilonproteobacteria and unidentified sequences obtained in this study, as well as from the GenBank database. The bootstrap values at the nodes are percentages based on 500 replicates. The names in brackets are those of well-known cold seeps and hydrothermal vents. Sequences from this study are indicated by bold type, and the numbers in parentheses indicate the numbers of sequences with 98% identity to the relevant sequences from the white mat, the sediment underlying the white mat, the orange mat, and the sediment underlying the orange mat. Only selected sequences are displayed. Sequences that are targeted by Arc94 and Mγ705 are indicated.

FIG. 9.

Maximum parsimony tree of 16S rRNA gene sequences from Archaea obtained in this study, as well as from the GenBank database. The names in brackets are those of well-known cold seeps and hydrothermal vents. The bootstrap values at the nodes are percentages based on 500 replicates. Sequences from this study are indicated by bold type, and the numbers in parentheses indicate the numbers of sequences with 98% identity to the relevant sequences from the white mat, the sediment underlying the white mat, the orange mat, and the sediment underlying the orange mat. Only selected sequences are displayed. The sequence with accession number DQ369741 was excluded from the bootstrap analysis and added to the tree using the parsimony tool in ARB. Sequences that are targeted by ANME-1-350, ANME-2-538, and ANME-3-1249 are indicated.