Formation of Multilayered Photosynthetic Biofilms in an Alkaline Thermal Spring in Yellowstone National Park,Wyoming

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°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.Biofilms are widely recognized as the way that most microbes exist in natural habitats (10-12), often developing into thick mats on various substrata associated with geysers and thermal springs in places such as Yellowstone National Park (53). In the first published microbiological studies of near-boiling pools in Yellowstone''s Lower Geyser Basin, an immersed glass slide approach was used to recover biofilm-forming microbial communities containing thermophilic Cyanobacteria, pink and yellow filaments, and many colorless rods (7, 8). Dispersal of microorganisms from biofilms has been investigated using 16S rRNA studies of geothermal vent source water and downstream travertine deposit samples at Angel Terrace, a carbonate spring in Mammoth Basin, Yellowstone National Park (18, 19). Using an artificial substrate and in situ kinetic apparatus at Angel Terrace, Kandianis et al. recently demonstrated that biomass serves as a catalyst in the precipitation of calcium carbonate (27). Regrowth of cyanobacterial mat communities after excision and removal at the alkaline Octopus Spring in the Lower Geyser Basin was investigated by Ferris et al. (16). In an effort to address how biofilms influence sinter formation, Cady and Farmer (9) observed that the presence of hyperthermophilic biofilms influenced the development of spicular geyserite by providing a preferred substratum for opaline silica precipitation. In a follow-up study, Blank et al. observed that communities in the subaqueous environment of alkaline, silica-depositing springs were similar to those associated with spicular geyserite at the air-water interface within the same spring (3). However, none of these studies has examined biofilm growth using artificial substrata to systematically evaluate community succession over time.Fairy Geyser represents a constantly erupting alkaline geothermal feature. Its vent waters (70 to 90°C) almost constantly erupt and splash, supporting the growth of extensive multilayered mat communities (35 to 60°C and pH 7.5 to 8.5) that form just above the water, on top of the sinter which surrounds the main geyser vent (5, 6). Fairy Geyser splash mats are composed of a green surface layer of Cyanobacteria and a red underlayer dominated by Roseiflexus-like Chloroflexi (5, 6). In monitoring Fairy Geyser since 1998, we have regularly observed the formation of similar multilayered photosynthetic microbial mats in splash zones at the cooler air-water interface (35 to 40°C) along the runoff channel or on debris that has fallen into the runoff channel (S. Boomer, unpublished results). Consequently, we hypothesized that solid substrata suspended in the Fairy Geyser runoff channel should support biofilm development and serve as a simple means for monitoring successional events over time, including the development of multilayered communities. Because Roseiflexus-like Chloroflexi from comparable red-layer communities in Yellowstone have been shown to be nonsulfur photoheterotrophs that metabolize under low-intensity light (6), we further hypothesized that red underlayer formation would require the presence of a shielding green layer and would thus form after initial colonization by Cyanobacteria. In addition to decreasing the light intensity, the photoautotrophic Cyanobacteria would provide organic compounds for the photoheterotrophic Chloroflexi, which typically consume cyanobacterial photosynthate in nature (40). Finally, we hypothesized that Roseiflexus-like Chloroflexi would be present in geothermal water at the vent source, providing an inoculum for substrata in the runoff channel.To test these hypotheses, we characterized the microbial community that accumulated at the air-water interface of sterile glass rods suspended in the thermal runoff at Fairy Geyser. Owing to environmental resource impact and winter access issues, this report encompasses two separate year-long studies. The first study (2004-2005) investigated the succession of the rod-associated communities during the first 3 months of colonization in the summer season, while the second study (2006-2007) investigated the succession after longer periods (6 to 8 months) of colonization during the winter season. In both cases, some replicates were maintained for 13 months, producing thick and gelatinous mat-like biofilms with outer green and inner red layering. For samples from all time points, we assessed biofilm accumulation by using microscopy, pigment analysis, and 16S rRNA studies, targeting both general bacteria and Chloroflexi. Although aforementioned Yellowstone biofilm studies have reported growing monolayers on artificial substrates or performed disturbance and recovery studies of existing mat systems in Yellowstone, this study represents the first report of multilayered photosynthetic mats being generated and studied in this manner.