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Applied and Environmental Microbiology, June 2003, p. 3607-3616, Vol. 69, No. 6
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.6.3607-3616.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Combining Culture-Dependent and -Independent Methodologies for Estimation of Richness of Estuarine Bacterioplankton Consuming Riverine Dissolved Organic Matter

Veljo Kisand{dagger} and Johan Wikner*

Department of Microbiology, Umeå University, SE-90187 Umeå, Sweden

Received 18 October 2002/ Accepted 12 March 2003

Three different methods for analyzing natural microbial community diversity were combined to maximize an estimate of the richness of bacterioplankton catabolizing riverine dissolved organic matter (RDOM). We also evaluated the ability of culture-dependent quantitative DNA-DNA hybridization, a 16S rRNA gene clone library, and denaturing gradient gel electrophoresis (DGGE) to detect bacterial taxa in the same sample. Forty-two different cultivatable strains were isolated from rich and poor solid media. In addition, 50 unique clones were obtained by cloning of the bacterial 16S rDNA gene amplified by PCR from the community DNA into an Escherichia coli vector. Twenty-three unique bands were sequenced from 12 DGGE profiles, excluding a composite fuzzy band of the Cytophaga-Flavobacterium group. The different methods gave similar distributions of taxa at the genus level and higher. However, the match at the species level among the methods was poor, and only one species was identified by all three methods. Consequently, all three methods identified unique subsets of bacterial species, amounting to a total richness of 97 operational taxonomic units in the experimental system. The confidence in the results was, however, dependent on the current precision of the phylogenetic determination and definition of the species. Bacterial consumers of RDOM in the studied estuary were primarily both cultivatable and uncultivable taxa of the Cytophaga-Flavobacterium group, a concordant result among the methods applied. Culture-independent methods also suggested several not-yet-cultivated ß-proteobacteria to be RDOM consumers.

* Corresponding author. Mailing address: Umeå Marine Sciences Center, Umeå University, Norrbyn SE-910 20, Hörnefors, Sweden. Phone: 46 (0)90 786 7980. Fax: 46 (0)90 786 7995. E-mail: johan.wikner{at}umf.umu.se.

{dagger} Present address: Võrtsjärv Limnological Station, EAU Institute of Zoology and Botany, Rannu EE-61101, Tartumaa, Estonia.

Applied and Environmental Microbiology, June 2003, p. 3607-3616, Vol. 69, No. 6
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.6.3607-3616.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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