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Applied and Environmental Microbiology, August 2006, p. 5596-5609, Vol. 72, No. 8
0099-2240/06/$08.00+0     doi:10.1128/AEM.00715-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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

Jennifer L. Macalady,1* Ezra H. Lyon,1 Bess Koffman,2 Lindsey K. Albertson,1 Katja Meyer,1 Sandro Galdenzi,3 and Sandro Mariani4

Department of Geosciences, Pennsylvania State University, University Park, Pennsylvania 16802,1 Geology Department, Carleton College, Northfield, Minnesota 55057,2 Istituto Italiano di Speleologia, Frasassi Section, Viale Verdi 10, 60035 Jesi, Italy,3 Gruppo Speleologico CAI, Via Alfieri 9, 60044 Fabriano, Italy4

Received 28 March 2006/ Accepted 14 June 2006

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. ß-, {gamma}-, {delta}-, and {varepsilon}-proteobacteria in sulfur-cycling clades accounted for ≥75% of clones in both biofilms. Sulfate-reducing and sulfur-disproportionating {delta}-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 {gamma}-proteobacteria with Beggiatoa-like and/or Thiothrix-like cells containing abundant sulfur inclusions. The biomass of {varepsilon}-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.

* Corresponding author. Mailing address: Geosciences Department, Pennsylvania State University, University Park, PA 16802. Phone: (814) 865-6330. Fax: (814) 863-7823. E-mail: jmacalad{at}geosc.psu.edu.

Applied and Environmental Microbiology, August 2006, p. 5596-5609, Vol. 72, No. 8
0099-2240/06/$08.00+0     doi:10.1128/AEM.00715-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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