Formation of multilayered photosynthetic biofilms in an alkaline thermal spring in Yellowstone National Park, Wyoming

Abstract

In this study, glass rods suspended at the air-water interface in the runoff channel of Fairy Geyser, Yellowstone National Park, WY, were used as a substratum to promote the development of biofilms that resembled multilayered mat communities in the splash zone at the geyser's source. This approach enabled the establishment of the temporal relationship between the appearance of Cyanobacteria, which ultimately formed the outer green layer, and the development of a red underlayer containing Roseiflexus-like Chloroflexi. This is the first study to define time-dependent successional events involved in the development of differently colored layers within microbial mats associated with many thermal features in Yellowstone National Park. Initial (1-month) biofilms were localized below the air-water interface (60 to 70 degrees C), and the majority of retrieved bacterial sequence types were similar to Synechococcus and Thermus isolates. Biofilms then shifted, becoming established at and above the air-water interface after 3 months. During winter sampling (6 to 8 months), distinct reddish orange microcolonies were observed, consistent with the appearance of Roseiflexus-like sequences and bacteriochlorophyll a pigment signatures. Additionally, populations of Cyanobacteria diversified to include both unicellular and filamentous cell and sequence types. Distinct green and red layers were observed at 13 months. Planctomycetes-like sequences were also retrieved in high abundance from final biofilm layers and winter samples. Finally, biomass associated with geyser vent water contained Roseiflexus-like sequence types, in addition to other high-abundance sequence types retrieved from biofilm samples, supporting the idea that geothermal water serves as an inoculum for these habitats.

FIG. 1.

Color plates showing macroscopic site and microscopic biofilm images. (A) Site images. Image a shows the natural splash mats located at the air-water interface by Fairy Geyser; the arrow indicates the community that has previously been sampled and described. Image b shows glass rods suspended in the Fairy Geyser runoff after 1 month (June 2004); the arrow indicates the air-water interface. Image c shows glass rods suspended in the Fairy Geyser runoff after 13 months (July 2007); the arrow indicates the biofilm samples growing at and above the air-water interface. (B) Macroscopic images of biofilm samples. Images a, b, c, and e show whole glass rod biofilm samples from 1 month (June 2004), 3 months (September 2004), 6 months (December 2006), and 8 months (February 2007), respectively. For images b, c, and e, arrows indicate the location of air-water interface at the time of collection. Image d shows the 13-month biofilm being homogenized during sample preparation; the arrow indicates notable reddish orange sheets that began to appear at this time point. Image f shows a biofilm sample at 6 months that has been removed from the glass rod to reveal a cross-section of the outer green and inner red layers; this entire sample grew at and above the air-water interface. (C) Microscopic images of biofilm and water samples. All top row images show samples viewed under transmitted light; all bottom row images show samples viewed under fluorescence using an Olympus U-MWIB2 filter set (excitation at 460 to 490 nm) to assess for red autofluorescence, indicative of Chl a; all bars measure 10 μm. Column a shows images from 3-month (September 2004) biofilm samples, which also represented the 1-month biofilm samples; the arrow indicates a nonfluorescing filament. Column b shows images from 6-month (December 2006) biofilm samples; the arrow indicates one of several notable reddish orange microcolonies, the filaments of which did not exhibit autofluorescence. Column c shows images from 8-month (February 2007) biofilm samples; the arrow indicates one of many filamentous cyanobacterial cell types that began to appear after 6 months. Column d shows images from 13-month green-layer samples; the arrow indicates a heterocyst along one of many filamentous cyanobacterial cell types in these samples. Column e shows images from 13-month red-layer samples, dominated by reddish orange filament masses that did not exhibit autofluorescence. Column f shows images from filtered water biomass; the arrow indicates a nonfluorescing filament.

FIG. 2.

Color plates showing graphs of pigments and bacterial retrieval rates. (A) Graphs showing pigment analysis and retrieval of phototrophs. In the top graph, total pigment was methanol extracted from each 0.02-g biofilm homogenate (for 1- to 8-month samples) and biofilm layer (for 13-month green and red layers). Absorbance was recorded for green Chl a or Bchl c pigment peaks (green bars) at 664 to 669 nm and for red pigment Bchl a peaks (red bars) at 768 to 771 nm. Pigments were quantified and reported in μg/g wet mat weight. In the bottom graph, the percentages of retrieved sequences for all Cyanobacteria-like sequences per general bacterial library (green), the percentages of retrieved sequences for all red Chloroflexi-like sequences per Chloroflexi library (maroon), and the percentages of retrieved sequences for all red Chloroflexi-like sequences per general bacterial library (red) are plotted over time. (B) Graph showing general-bacterial-library analysis, with rarefaction and abundance data. In the top graph, rarefaction curves for total bacterial phyla are shown for each of the assessed water and biofilm samples; coverage values (C) for each sample are indicated in the legend. In the bottom graph, the percentages of retrieved sequences for all phyla from general bacterial libraries are shown for water and biofilm samples over time; each phylum has been color coded according to the key shown adjacent to the graph.

FIG. 3.

Maximum parsimony tree of Chloroflexi library clones. Known Chloroflexi are indicated in italics, with the GenBank accession numbers in parentheses. Representative water and biofilm clones are signified by bold clone names and numbers (Table 4). The bar represents 10 nucleotide changes. In this analysis, there were 1,000 total characters, and 370 were usable for parsimony analysis. A total of 100 bootstrap replicates were performed, and the bootstrap values are indicated (those that were <50% are not shown).