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. 2018 May 22:9:796.
doi: 10.3389/fmicb.2018.00796. eCollection 2018.

Key Role of Alphaproteobacteria and Cyanobacteria in the Formation of Stromatolites of Lake Dziani Dzaha (Mayotte, Western Indian Ocean)

Emmanuelle Gérard  1 , Siham De Goeyse  1 , Mylène Hugoni  2 , Hélène Agogué  3 , Laurent Richard  4 , Vincent Milesi  1 , François Guyot  5 , Léna Lecourt  1 , Stephan Borensztajn  1 , Marie-Béatrice Joseph  1 , Thomas Leclerc  1 , Gérard Sarazin  1 , Didier Jézéquel  1 , Christophe Leboulanger  6 , Magali Ader  1
Affiliations

Key Role of Alphaproteobacteria and Cyanobacteria in the Formation of Stromatolites of Lake Dziani Dzaha (Mayotte, Western Indian Ocean)

Emmanuelle Gérard et al. Front Microbiol. .

Abstract

Lake Dziani Dzaha is a thalassohaline tropical crater lake located on the "Petite Terre" Island of Mayotte (Comoros archipelago, Western Indian Ocean). Stromatolites are actively growing in the shallow waters of the lake shores. These stromatolites are mainly composed of aragonite with lesser proportions of hydromagnesite, calcite, dolomite, and phyllosilicates. They are morphologically and texturally diverse ranging from tabular covered by a cauliflower-like crust to columnar ones with a smooth surface. High-throughput sequencing of bacterial and archaeal 16S rRNA genes combined with confocal laser scanning microscopy (CLSM) analysis revealed that the microbial composition of the mats associated with the stromatolites was clearly distinct from that of the Arthrospira-dominated lake water. Unicellular-colonial Cyanobacteria belonging to the Xenococcus genus of the Pleurocapsales order were detected in the cauliflower crust mats, whereas filamentous Cyanobacteria belonging to the Leptolyngbya genus were found in the smooth surface mats. Observations using CLSM, scanning electron microscopy (SEM) and Raman spectroscopy indicated that the cauliflower texture consists of laminations of aragonite, magnesium-silicate phase and hydromagnesite. The associated microbial mat, as confirmed by laser microdissection and whole-genome amplification (WGA), is composed of Pleurocapsales coated by abundant filamentous and coccoid Alphaproteobacteria. These phototrophic Alphaproteobacteria promote the precipitation of aragonite in which they become incrusted. In contrast, the Pleurocapsales are not calcifying but instead accumulate silicon and magnesium in their sheaths, which may be responsible for the formation of the Mg-silicate phase found in the cauliflower crust. We therefore propose that Pleurocapsales and Alphaproteobacteria are involved in the formation of two distinct mineral phases present in the cauliflower texture: Mg-silicate and aragonite, respectively. These results point out the role of phototrophic Alphaproteobacteria in the formation of stromatolites, which may open new perspective for the analysis of the fossil record.

Keywords: Alphaproteobacteria; Mg-silicate; Pleurocapsales; alkaline lake; anoxygenic phototrophic bacteria; aragonite; hydromagnesite; stromatolites.

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Figures

FIGURE 1
FIGURE 1
(A) Aerial view of the Dziani Dzaha Lake in October 2014 showing the positions of the sampled stromatolites (M6–M19) and the points where water was collected (point 18 m) for the chemical analysis. A general description of the types of the different stromatolites found on the North (8), South (2), West (4), and East (9) shores is indicated in the red boxes. No stromatolites or only scarce friable flat crusts were found on the East shore. Cr and Fr describe the surface of stromatolites, Cr: crust, Fr: friable. (B) Global view of the lake with clumps of emerged columnar stromatolites on the south part of the lake. The green color of the water is due to the high abundance of Cyanobacteria. The purple color visible on the shore may be related to purple bacteria. (C) A columnar stromatolite (M6) collected 30 cm under the water surface covered by a greenish to purple microbial mat. (D) A columnar stromatolite (M16), collected 20 cm under the water surface with a cauliflower-like texture covered by a purple to green thin biofilm. Note that both stromatolites are partly covered by fly pupae except the top few centimeters of the M16 stromatolite that was probably formed after the last fly laying.
FIGURE 2
FIGURE 2
Confocal laser scanning microscopy (CLSM) and optical images of the microbial mats associated with the cauliflower-like crust (A,C,F) or columnar stromatolites with friable surfaces (B,D,E,G). (A) Pleurocapsales (red) coated by filamentous microorganisms (green) located at the surface of the microbial mat associated with M17 fixed with formaldehyde and ethanol and stained with Syto9® (green) observed with CLSM. (B) Filamentous Cyanobacteria (red) with persistent sheath (blue) detected by CLSM in the microbial mat associated with M12 fixed with formaldehyde and ethanol and stained with Syto9® (the Cyanobacteria detected in M15 had the same morphology as in M12). (C) Microbial mat associated with M17 fixed with RNAlater®, stained with Syto9® and observed with CLSM where micrometric coccoid microorganisms are visible in the empty sheath of Pleurocapsales (white star) and in association with refractive dots (blue, white arrow) which are probably elemental sulfur globules produced by the coccoid microorganisms. (D) Filamentous Cyanobacteria detected in the smooth surface of M10 which arbor shorter trichomes than Cyanobacteria detected on M12 and M15. (F) Microbial mat associated with M17 fixed with RNAlater and observed with optical microscopy in which elemental sulfur grains (black arrow) associated with the purple bacteria located around the Pleurocapsales cells (black star) are visible. (E) Red microbial mat associated with M15 fixed with RNA later and stained with Syto9® in which refractive grains (blue, white arrow) in rod shaped bacteria probably corresponding to elemental sulfur globules are visible. (G) Transversal cutting of M10 stained with DAPI (blue) and calcein (green), included in LR white resin and observed with CLSM where aragonite grains stained by calcein (white arrow) trapped by filamentous Cyanobacteria stained with DAPI are visible. (H) Colors associated with the CLSM images.
FIGURE 3
FIGURE 3
(A) Relative abundance of the different cyanobacterial classes inferred from 16S rRNA gene sequences in Dziani Dzaha stromatolites. (B) Proportion of sequences affiliated with major bacterial phyla (defined as >0.2% of total sequence number). (C) Relative abundance of the different alphaproteobacterial orders inferred from 16S rRNA genes sequences in Dziani Dzaha stromatolites.
FIGURE 4
FIGURE 4
Scanning electron microscopy (SEM) images of a transversal cutting of the cauliflower-like crust from a pink colored area of M17, embedded in LR white resin, showing the mineral distribution in the microbial mat. (A) Relics of micrometric cells (white arrow) that seemingly served as nucleation points for aragonite (bright gray) precipitation. (B) Hydromagnesite needles (dark gray, white arrow) growing on aragonite grains.
FIGURE 5
FIGURE 5
Scanning electron microscopy images of a transversal cutting of the cauliflower-like crust from a green/pink colored area of M17, embedded in LR white resin (A–F), or top of the microbial mat of M17 desiccated to the critical point (G,H), showing the mineral distribution in the microbial mat. In all the images red stars correspond to aragonite; green stars correspond to hydromagnesite; blue stars to magnesium silicate. (A) Global view of the globular crust (at 15 KV, AsB detector), bright gray indicates the Ca rich phases and dark gray the Mg rich phases. Colonies of Pleurocapsales are visible on the top of the transversal cutting. (B–F) Enlargement of the transversal cutting shown in (A). (B) Aragonite zone at the bottom of the transversal cutting with relics of micrometric cells (at 15 KV, AsB detector). (C) Hydromagnesite needles grown on aragonite (at 15 KV, AsB detector). (D) Aragonite grown on relics of Pleurocapsales cells embedded in a magnesium silicate phase (at 15 KV, AsB detector). (E) Relics of Pleurocapsales colonies embedded in a magnesium silicate phase (at 15 KV, AsB detector). (F) Aragonite precipitated at the bottom of Pleurocapsales colonies and hydromagnesite needles on aragonite (at 15 KV, AsB detector). (G) Aragonite precipitated around Pleurocapsales cells at the surface of the microbial mat (at 10 KV, AsB detector). (H) Pleurocapsales embedded in a magnesium silicate phase (at 10 KV, AsB detector). (H-a – H-c) Enlargements of the SEM image shown in (H) highlighting Mg-Si beads around the Pleurocapsales cells. White arrow in (H-b) indicates holes in the silicate magnesium phase. White arrows in (H-c) indicate silicate beads around Pleurocapsales cells.
FIGURE 6
FIGURE 6
Scanning electron microscopy-EDS/CLSM/Raman on the same transversal cutting of the globular crust of M16 embedded in LR white resin, showing the accumulation of silicon in the sheaths of Pleurocapsales. (A,B) SEM-EDS mappings of calcium, magnesium, and silicon. (C) SEM-EDS mapping of calcium and silicon in the same area as in (A,B). (B,C) Are enlargement of the mapping shown in (A). The white rectangle shows exactly the same zone in the four images (A–D). In (A–C), the EDS mapping shows that calcium and silicon were negatively correlated whereas magnesium and silicon were sometimes positively correlated. (D) CLSM image of the same area showing that the silicon accumulated in the empty sheaths of Pleurocapsales. (E) Raman spectra of points a, b, c, d, and e highlighted in (D). The vertical line fits the specific peak for hydromagnesite detected at points a, e, and c. The other peaks are specific for aragonite that was detected at all points. (F) Colors associated with the CLSM (D) and EDS mappings (A–C).
FIGURE 7
FIGURE 7
(A) Confocal laser scanning microscopy image of a transversal cutting of the globular crust of M17 embedded in LR white resin, showing the Pleurocapsales colonies (pink/red) topped with grains stained with calcein (green) and filamentous Alphaproteobacteria also strongly stained with calcein (green, white star). (B) Raman mapping superimposed on the CLSM image showing the distribution of the aragonite grains around the Pleurocapsales colonies. (C) Colors associated with the CLSM and Raman mappings. (D) CLSM image of the microbial mat fixed in RNAlater and stained with Syto9 (green) showing the distribution of the Pleurocapsales colonies topped with coccoid microorganisms associated with aragonite grains and filamentous cells on the top. (E) SEM image (at 15 KV, AsB detector) on the same transversal cutting shown in (A,B), showing aragonite grains associated with the coccoid and filamentous microorganisms associated with the Pleurocapsales. Red stars correspond to aragonite and the green star to hydromagnesite in all images.
FIGURE 8
FIGURE 8
Confocal laser scanning microscopy/Raman/SEM-EDS on the same transversal cutting in red colored zone of M12 microbial mat embedded in LR white showing aragonite precipitating around the microbial mat. (A) CLSM image showing coccoid, rod shaped and filamentous microorganisms (blue) being incrusted in aragonite (green). No Cyanobacteria were detected in this zone. (B) Colors associated with the CLSM and EDS mappings. (C) EDS mapping of the same transversal cutting showing the centers of aragonite grains where magnesium and phosphorus is detected, which could correspond to relics of the encrusted microbial mat. (D) SEM image of the same image as in (C) (at 15 KV, AsB detector). The LR white resin appears in black and aragonite in bright gray.
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