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Applied and Environmental Microbiology, April 2004, p. 2230-2239, Vol. 70, No. 4
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.4.2230-2239.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Survey of Archaeal Diversity Reveals an Abundance of Halophilic Archaea in a Low-Salt, Sulfide- and Sulfur-Rich Spring

Mostafa S. Elshahed,1 Fares Z. Najar,2 Bruce A. Roe,2 Aharon Oren,3 Thomas A. Dewers,4 and Lee R. Krumholz1*

Department of Botany and Microbiology and Institute for Energy and the Environment,1 Department of Chemistry and Biochemistry,2 Department of Geology and Geophysics, University of Oklahoma, Norman, Oklahoma 73019,4 Institute of Life Sciences and Moshe Shilo Minerva Center for Marine Biogeochemistry, The Hebrew University of Jerusalem, Jerusalem, Israel3

Received 3 October 2003/ Accepted 12 December 2003

The archaeal community in a sulfide- and sulfur-rich spring with a stream water salinity of 0.7 to 1.0% in southwestern Oklahoma was studied by cloning and sequencing of 16S rRNA genes. Two clone libraries were constructed from sediments obtained at the hydrocarbon-exposed source of the spring and the microbial mats underlying the water flowing from the spring source. Analysis of 113 clones from the source library and 65 clones from the mat library revealed that the majority of clones belonged to the kingdom Euryarchaeota, while Crenarchaeota represented less than 10% of clones. Euryarchaeotal clones belonged to the orders Methanomicrobiales, Methanosarcinales, and Halobacteriales, as well as several previously described lineages with no pure-culture representatives. Those within the Halobacteriales represented 36% of the mat library and 4% of the source library. All cultivated members of this order are obligately aerobic halophiles. The majority of halobacterial clones encountered were not affiliated with any of the currently described genera of the family Halobacteriaceae. Measurement of the salinity at various locations at the spring, as well as along vertical gradients, revealed that soils adjacent to spring mats have a much higher salinity (NaCl concentrations as high as 32%) and a lower moisture content than the spring water, presumably due to evaporation. By use of a high-salt-plus-antibiotic medium, several halobacterial isolates were obtained from the microbial mats. Analysis of 16S rRNA genes indicated that all the isolates were members of the genus Haloferax. All isolates obtained grew at a wide range of salt concentrations, ranging from 6% to saturation, and all were able to reduce elemental sulfur to sulfide. We reason that the unexpected abundance of halophilic Archaea in such a low-salt, highly reduced environment could be explained by their relatively low salt requirement, which could be satisfied in specific locations of the shallow spring via evaporation, and their ability to grow under the prevalent anaerobic conditions in the spring, utilizing zero-valent sulfur compounds as electron acceptors. This study demonstrates that members of the Halobacteriales are not restricted to their typical high-salt habitats, and we propose a role for the Halobacteriales in sulfur reduction in natural ecosystems.

* Corresponding author. Mailing address: University of Oklahoma, Department of Botany and Microbiology, 770 Van Vleet Oval, Norman, OK 73019. Phone: (405) 325-0437. Fax: (405) 325-7619. E-mail: Krumholz{at}ou.edu.

Applied and Environmental Microbiology, April 2004, p. 2230-2239, Vol. 70, No. 4
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.4.2230-2239.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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