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Applied and Environmental Microbiology, December 2004, p. 7378-7387, Vol. 70, No. 12
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.12.7378-7387.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Bacterial Diversity in a Nonsaline Alkaline Environment: Heterotrophic Aerobic Populations

Igor Tiago, Ana Paula Chung, and António Veríssimo*

Departamento de Zoologia and Centro de Neurociências, Universidade de Coimbra, Coimbra, Portugal

Received 23 March 2004/ Accepted 16 August 2004

Heterotrophic populations were isolated and characterized from an alkaline groundwater environment generated by active serpentinization, which results in a Ca(OH)2-enriched, extremely diluted groundwater with pH 11.4. One hundred eighty-five strains were isolated in different media at different pH values during two sampling periods. To assess the degree of diversity present in the environment and to select representative strains for further characterization of the populations, we screened the isolates by using random amplified polymorphic DNA-PCR profiles and grouped them based on similarities determined by fatty acid methyl ester analysis. Phenotypic characterization, determinations of G+C content, phylogenetic analyses by direct sequencing of 16S rRNA genes, and determinations of pH tolerance were performed with the selected isolates. Although 38 different populations were identified and characterized, the vast majority of the isolates were gram positive with high G+C contents and were affiliated with three distinct groups, namely, strains closely related to the species Dietzia natrolimnae (32% of the isolates), to Frigoribacterium/Clavibacter lineages (29% of the isolates), and to the type strain of Microbacterium kitamiense (20% of the isolates). Other isolates were phylogenetically related to strains of the genera Agrococcus, Leifsonia, Kytococcus, Janibacter, Kocuria, Rothia, Nesterenkonia, Citrococcus, Micrococcus, Actinomyces, Rhodococcus, Bacillus, and Staphylococcus. Only five isolates were gram negative: one was related to the Sphingobacteria lineage and the other four were related to the {alpha}-Proteobacteria lineage. Despite the pH of the environment, the vast majority of the populations were alkali tolerant, and only two strains were able to grow at pH 11.

* Corresponding author. Mailing address: Departamento de Zoologia, Universidade de Coimbra, 3004-517 Coimbra, Portugal. Phone: 351 239824024. Fax: 351 239826798. E-mail: averiss{at}ci.uc.pt.

Applied and Environmental Microbiology, December 2004, p. 7378-7387, Vol. 70, No. 12
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.12.7378-7387.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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