Dominant Microbial Populations in Limestone-Corroding Stream Biofilms, Frasassi Cave System, Italy

Abstract

Waters from an extensive sulfide-rich aquifer emerge in the Frasassi cave system, where they mix with oxygen-rich percolating water and cave air over a large surface area. The actively forming cave complex hosts a microbial community, including conspicuous white biofilms coating surfaces in cave streams, that is isolated from surface sources of C and N. Two distinct biofilm morphologies were observed in the streams over a 4-year period. Bacterial 16S rDNA libraries were constructed from samples of each biofilm type collected from Grotta Sulfurea in 2002. ␤-, ␥-, ␦-, and -proteobacteria in sulfur-cycling clades accounted for >75% of clones in both biofilms. Sulfate-reducing and sulfur-disproportionating ␦-proteobacterial sequences in the clone libraries were abundant and diverse (34% of phylotypes). Biofilm samples of both types were later collected at the same location and at an additional sample site in Ramo Sulfureo and examined, using fluorescence in situ hybridization (FISH). The biomass of all six stream biofilms was dominated by filamentous ␥-proteobacteria with Beggiatoa-like and/or Thiothrix-like cells containing abundant sulfur inclusions. The biomass of -proteobacteria detected using FISH was consistently small, ranging from 0 to less than 15% of the total biomass. Our results suggest that S cycling within the stream biofilms is an important feature of the cave biogeochemistry. Such cycling represents positive biological feedback to sulfuric acid speleogenesis and related processes that create subsurface porosity in carbonate rocks.Sulfidic caves form in carbonate rocks where sulfide-rich waters interact with oxygen at the water table or at subterranean springs. The caves form as a result of sulfuric acid production (equation 1) from microbial or abiotic sulfur oxidation. The sulfuric acid reacts with carbonate host rock to form gypsum and carbonic acid (equation 2).(1)Some of the longest caves known are thought to have formed by this process, including Lechugilla Cave in New Mexico, with 184 km of passages (17). Actively forming sulfidic caves are uncommon but intensely valuable as natural laboratories to understand factors influencing cave formation and resulting biological, geochemical, and isotopic signatures. Active sulfidic caves can host biogeochemically isolated ecosystems based entirely on microbial lithoautotrophic primary productivity (16,38). These ecosystems are aphotic, terrestrial, subsurface environments comparable to sulfureta at hot springs and deep sea vents (10) and are of considerable interest as analogs for microbially dominated, early earth biotic communities such as those that might have developed after the initial rise of oxygen in the early Proterozoic era.Available information from culturing, fluorescence in situ hybridization (FISH), and 16S rDNA libraries suggests that ε-and ␥-proteobacteria are important biofilm-forming groups in the sulfidic cave waters studied to date. Microbial biofilms in Lower Kane Cave (Wyoming) springs and streams are dominated by filamentous ε-prot...

Bacterial Diversity and Composition in Oylat Cave (Turkey) with Combined Sanger/Pyrosequencing Approach

“…For instance, Nitrospirae clones observed from the extremely acidic Frasassi Cave (Macalady et al, 2006). Members of Nitrospirae also observed in the limestone caves, Pajsarjeva jama Cave and Tito Bustillo Cave (Schabereiter-Gurtner et al, 2002;Pasic et al, 2010).…”

Isotopic evidence of enhanced carbonate dissolution at a coal mine drainage site in Allegheny County, Pennsylvania, USA

“…The eubacterial probe mix EUB338-I, -II, -III was used for detection of all Bacteria (Daims et al, 1999). The DELTA495A probe (Loy et al, 2002), applied with its competitor probe (Macalady et al, 2006), was used to detect the Deltaproteobacteria. Probes were applied at 50 ng/lL in 25% formamide.…”

A novel symbiosis between chemoautotrophic bacteria and a freshwater cave amphipod

“…The gnathopods of N. ictus, which are claw-like appendages used in amphipod species for grooming, are covered with a dense growth of Thiothrix filaments (Supplementary Figure 3). Moreover, Thiothrix filaments attached to N. ictus typically appear to be trimmed to a short and uniform length (Supplementary Figure 7) compared with biofilm-forming Thiothrix filaments we observe in cave streams (Macalady et al, 2006(Macalady et al, , 2008. Thus, it is likely that the Thiothrix epibionts form at least one component of the N. ictus diet.…”

“…For a detailed description of the geochemistry and microbial communities within the cave system, please refer to Macalady et al (2008) and Macalady et al (2006) Supplementary Figure 2 for map of the Frasassi cave system and location of collection sites). Samples for FISH and clone library construction were collected into sterile tubes, stored on ice, and transferred to four parts RNAlater (Ambion/ Applied Biosystems, Foster City, CA, USA) to one part sample (v/v) within 4-6 h after collection, and stored at À20 1C until further analysis.…”

“…Conspicuous matlike white biofilms cover surfaces near the water table where sulfidic and oxygenated waters mix, and where sulfidic springs contact the oxygenated cave air (Supplementary Figure 1). Detailed studies conducted earlier by our group revealed that the biofilms are predominantly composed of sulfurcycling bacteria within the b-, d-, g-, and e-proteobacterial clades (Macalady et al, 2006). Specifically, filamentous Thiothrix, Beggiatoa, and e-proteobacteria dominate the biomass of microbial biofilms, inhabiting separate niches defined by water chemistry and stream flow characteristics (Macalady et al, 2008).…”