A Study of the Microbial Spatial Heterogeneity of Bahamian Thrombolites Using Molecular, Biochemical, and Stable Isotope Analyses

Abstract

Thrombolites are buildups of carbonate that exhibit a clotted internal structure formed through the interactions of microbial mats and their environment. Despite recent advances, we are only beginning to understand the microbial and molecular processes associated with their formation. In this study, a spatial profile of the microbial and metabolic diversity of thrombolite-forming mats of Highborne Cay, The Bahamas, was generated by using 16S rRNA gene sequencing and predictive metagenomic analyses. These molecular-based approaches were complemented with microelectrode profiling and in situ stable isotope analysis to examine the dominant taxa and metabolic activities within the thrombolite-forming communities. Analyses revealed three distinctive zones within the thrombolite-forming mats that exhibited stratified populations of bacteria and archaea. Predictive metagenomics also revealed vertical profiles of metabolic capabilities, such as photosynthesis and carboxylic and fatty acid synthesis within the mats that had not been previously observed. The carbonate precipitates within the thrombolite-forming mats exhibited isotopic geochemical signatures suggesting that the precipitation within the Bahamian thrombolites is photosynthetically induced. Together, this study provides the first look at the spatial organization of the microbial populations within Bahamian thrombolites and enables the distribution of microbes to be correlated with their activities within modern thrombolite systems. Key Words: Thrombolites-Microbial diversity-Metagenome-Stable isotopes-Microbialites. Astrobiology 17, 413-430.

FIG. 1

The thrombolites of Highborne Cay, The Bahamas. (A) Intertidal thrombolite platforms from Site 5. Bar, 1 m. (B) Light micrograph of a thrombolite-forming button mat revealing extensive vertical assemblages of calcified filaments (arrows). Bar, 500 μm. (C) In situ depth profiles of oxygen (square), sulfide (triangle), and pH (circle) collected at peak of photosynthesis (open symbols) or respiration (filled symbols). Shaded areas reflect the targeted areas collected for analysis. Depths below 9 cm were not sampled, as that region shared the same biochemical profile as in Zone 3. (D) Cross section of button mat depicting the three spatial regions including an oxic Zone 1 (0–3 mm), transitional Zone 2 (3–5 mm), and anoxic Zone 3 (5–9 mm). Bar, 3 mm.

FIG. 2

Taxonomic distribution of cyanobacteria within the thrombolite-forming mats derived from MEGAN5 using the Greengenes database. Overall percentages based on read counts are presented logarithmically, depicting the distributions for Zone 1 (blue), Zone 2 (green), and Zone 3 (red). Read abundance data for each taxonomic level are included in parentheses.

FIG. 3

Taxonomic distribution of Bacteria within the thrombolite-forming mats derived from MEGAN5 using the Greengenes database. Overall percentages based on read counts are presented logarithmically, depicting the distributions for Zone 1 (blue), Zone 2 (green), and Zone 3 (red). Read abundance data for each taxonomic level are included in parentheses.

FIG. 4

Comparison of diversity analyses of three spatial zones within the thrombolite-forming mats. Principal coordinate analysis of communities from unweighted UniFrac distance matrix of Zone 1 (0–3 mm, blue), Zone 2 (3–5 mm, green), and Zone 3 (5–9 mm, red) in (A) Bacteria and (B) Archaea populations. Ellipses represent standard deviation over 10 rarefaction samplings. Adonis tests suggest that depth is a significant predictor of community composition for both bacterial (R = 0.402, p = 0.001) and archaeal (R = 0.307, p = 0.017) communities.

FIG. 5

Taxonomic distribution of Archaea within the thrombolite-forming mats derived from MEGAN5 using the Greengenes database. Overall percentages based on read counts are presented logarithmically, depicting the distributions for Zone 1 (blue), Zone 2 (green), and Zone 3 (red). Read abundance data for each taxonomic level are included in parentheses.

FIG. 6

Functional gene comparison of the three thrombolitic mat spatial zones from 16S rRNA metabolic prediction (PICRUSt) and whole shotgun sequencing. Pearson correlation value (r) is shown for the comparison of metabolic predictions for Zone 1 (blue), Zone 2 (green), and Zone 3 (red) and the whole mat shotgun metagenome.

FIG. 7

Stable isotope results for calcified filaments located in the upper 3 mm of thrombolite-forming button mat. (A) Oxygen isotope values of organic and inorganic fractions using both bulk and SIMS analysis. Analyses were completed for both background carbonate precipitates (sediment), calcified filaments (filaments), and untreated whole mat samples. (B) Carbon and nitrogen isotope values of both organic and inorganic fractions using both bulk and SIMS analysis. (C) Comparative plot of SIMS values collected for oxygen and carbon isotopes. All results are expressed in delta notation with respect to the carbon/oxygen Vienna Pee Dee Belemnite (VPDB) or nitrogen air (AIR) standard.

FIG. 8

Overview of target areas for SIMS analyses within the thrombolite-forming mat. (A) Petrographic thin section of Dichothrix sp. filaments (f) and associated carbonate precipitate (cp) surrounded by sediments such as ooids (o). (B) Gold-coated reflected light image as viewed by the SIMS instrument. (C) Scanning electron micrograph showing the numerous 6–10 μm pits formed during the SIMS analysis. Boxes depict representative pits that show both high (green) and low (red) quality targets within the sample. (D) Higher-resolution scanning electron micrograph of representative high-quality pit (corresponding to green box in C) showing no textural anomalies or cracks. (E) Scanning electron micrograph of low-quality pit (corresponding to red box in C) showing crack within the targeted sample site. All low-quality target sites were removed from downstream analyses.