RESEARCH

Initial report on microbial stalactites and other biofilms from Manantial del Toro

(Macalady et al. 2010, unpublished)

Samples of microbial stalactites and associated microbial biofilms were collected in sterile containers by Phillip Lehman in May 2009. According to Phillip, these are fast-growing biofilms that colonize and produce obvious growth on dive line over a period of weeks. The samples were preserved on site in RNAlater and shipped to the Macalady Lab at Pennsylvania State University. Irene Schaperdoth in the Macalady Lab extracted DNA from the samples and made libraries of 16S rRNA taxonomic marker genes using PCR primers for the Archaea and Bacteria domains. Both libraries were successful after some troubleshooting to remove biofilm components that inhibited the PCR reactions.

The pie charts below summarize the taxonomic composition of Bacteria (86 clones) and Archaea (78 clones) in the entrance floor biofilm material. The taxonomic groups were assigned based on phylogenetic trees made using the quick-add parsimony function of the software package ARB.

Several features of these libraries are worth a comment. First, the great majority of the sequences belong to clades with no cultivated representatives, meaning that we cannot guess anything about their metabolism without further study. These sequences represent potentially novel organisms. Second, the few clones that do have close relationships to more familiar groups probably make their living by cycling nitrogen (e.g., relatives of the genera Scalindua , Nitrosopumulis , and Nitrospira ). Lastly, no clones related to known sulfate reducing bacteria were retrieved.

A preliminary interpretation of these data is that the microbial stalactites are composed of anaerobic and microaerophilic bacteria and archaea that produce organic matter from CO2 using energy derived from aerobic and anaerobic ammonia oxidation. The lack of sulfate reducing bacteria is consistent with diver observations that the waters do not have a hydrogen sulfide smell. The absence of sulfate reduction is notable, because the waters are reported to be 30-100% marine salininty, and therefore have abundant sulfate.

Reevaluation of this scenario, and a fuller understanding of the microbiology and geochemistry of the Manantial del Toro system, will be obtained following a science expedition planned for 2011. Stay tuned....