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Applied and Environmental Microbiology, April 2006, p. 2637-2643, Vol. 72, No. 4
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.4.2637-2643.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Cultivation of Denitrifying Bacteria: Optimization of Isolation Conditions and Diversity Study{dagger}

Kim Heylen,1* Bram Vanparys,1 Lieven Wittebolle,2 Willy Verstraete,2 Nico Boon,2 and Paul De Vos1

Laboratory of Microbiology, Department of Biochemistry, Physiology and Microbiology, Ghent University, K.L. Ledeganckstraat 35, B-9000 Ghent, Belgium,1 Laboratory of Microbial Ecology and Technology (LabMET), Ghent University, Coupure Links 653, B-9000 Ghent, Belgium2

Received 6 December 2005/ Accepted 30 January 2006

An evolutionary algorithm was applied to study the complex interactions between medium parameters and their effects on the isolation of denitrifying bacteria, both in number and in diversity. Growth media with a pH of 7 and a nitrogen concentration of 3 mM, supplemented with 1 ml of vitamin solution but not with sodium chloride or riboflavin, were the most successful for the isolation of denitrifiers from activated sludge. The use of ethanol or succinate as a carbon source and a molar C/N ratio of 2.5, 20, or 25 were also favorable. After testing of 60 different medium parameter combinations and comparison with each other as well as with the standard medium Trypticase soy agar supplemented with nitrate, three growth media were highly suitable for the cultivation of denitrifying bacteria. All evaluated isolation conditions were used to study the cultivable denitrifier diversity of activated sludge from a municipal wastewater treatment plant. One hundred ninety-nine denitrifiers were isolated, the majority of which belonged to the Betaproteobacteria (50.4%) and the Alphaproteobacteria (36.8%). Representatives of Gammaproteobacteria (5.6%), Epsilonproteobacteria (2%), and Firmicutes (4%) and one isolate of the Bacteroidetes were also found. This study revealed a much more diverse denitrifying community than that previously described in cultivation-dependent research on activated sludge.

* Corresponding author. Mailing address: Laboratory of Microbiology, Department of Biochemistry, Physiology and Microbiology, Ghent University, K.L. Ledeganckstraat 35, B-9000 Ghent, Belgium. Phone: 32(0)92645101. Fax: 32(0)92645092. E-mail: Kim.Heylen{at}UGent.be.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

Applied and Environmental Microbiology, April 2006, p. 2637-2643, Vol. 72, No. 4
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.4.2637-2643.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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