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Applied and Environmental Microbiology, July 2005, p. 3624-3632, Vol. 71, No. 7
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.7.3624-3632.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Diversity of Bacteria Growing in Natural Mineral Water after Bottling

Alexander Loy,1,3* Wolfgang Beisker,3 and Harald Meier2,3

Abteilung für Mikrobielle Ökologie, Institut für Ökologie und Naturschutz, Universität Wien, A-1090 Vienna, Austria,1 Angewandte Bioinformatik, Lehrstuhl für Rechnertechnik und Rechnerorganisation, Technische Universität München, D-80290 Munich, Germany,2 Arbeitsgruppe für Durchflusszytometrie, GSF-Forschungszentrum für Umwelt und Gesundheit, Ingolstädter Landstrasse 1, D-85764 Neuherberg, Germany3

Received 2 December 2004/ Accepted 27 January 2005

Bacterial growth occurs in noncarbonated natural mineral waters a few days after filling and storage at room temperature, a phenomenon known for more than 40 years. Using the full-cycle rRNA approach, we monitored the development of the planktonic bacterial community in a noncarbonated natural mineral water after bottling. Seven 16S rRNA gene libraries, comprising 108 clones in total, were constructed from water samples taken at various days after bottling and from two different bottle sizes. Sequence analyses identified 11 operational taxonomic units (OTUs), all but one affiliated with the betaproteobacterial order Burkholderiales (6 OTUs) or the class Alphaproteobacteria (4 OTUs). Fluorescence in situ hybridization (FISH) was applied in combination with DAPI (4',6'-diamidino-2-phenylindole) staining, viability staining, and microscopic counting to quantitatively monitor changes in bacterial community composition. A growth curve similar to that of a bacterium grown in a batch culture was recorded. In contrast to the current perception that Gammaproteobacteria are the most important bacterial components of natural mineral water in bottles, Betaproteobacteria dominated the growing bacterial community and accounted for 80 to 98% of all bacteria detected by FISH in the late-exponential and stationary-growth phases. Using previously published and newly designed genus-specific probes, members of the betaproteobacterial genera Hydrogenophaga, Aquabacterium, and Polaromonas were found to constitute a significant proportion of the bacterial flora (21 to 86% of all bacteria detected by FISH). For the first time, key genera responsible for bacterial growth in a natural mineral water were identified by applying molecular cultivation-independent techniques.

* Corresponding author. Mailing address: Abteilung für Mikrobielle Ökologie, Institut für Ökologie und Naturschutz, Universität Wien, Althanstr. 14, A-1090 Vienna, Austria. Phone: 43-1-4277-54207. Fax: 43-1-4277-54389. E-mail: loy{at}microbial-ecology.net.

Applied and Environmental Microbiology, July 2005, p. 3624-3632, Vol. 71, No. 7
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.7.3624-3632.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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