Cultivation of Denitrifying Bacteria: Optimization of Isolation Conditions and Diversity Study

Abstract

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.For nearly 2 decades, molecular biology has provided the tools to successfully overcome the "great plate count anomaly" and allow the study of uncultured microbial diversity (3). The growing awareness that molecular methods cannot or, in very few cases, can only indirectly investigate the function of specific microorganisms in the environment has raised interest in new cultivation efforts and approaches once again (14,15,34). Simple adjustments to the classical cultivation approach, such as prolonging the incubation time and avoiding complex or nutrient-rich growth media, have successfully resulted in cultivation of previously uncultured bacteria (12,30).A physiological trait such as denitrification, the respiratory reduction of nitrate and nitrite to N 2 O and nitrogen gas, is not limited to specific microbial taxa and is therefore studied independent of culture through the relevant functional genes (6,25,32,38). To date, however, it is not clear to what extent, if at all, these functional genes contain phylogenetic information. Phillipot (22) showed that the phylogeny of nir and nor genes, coding for the key enzymes nitrite reductase and NO reductase in the denitrification pathway, does not always agree with the phylogeny of the 16S rRNA gene. New isolation and cultivation approaches are therefore imperative to provide the basis for further research on phylogenetic and functional gene diversity.The isolation of specific physiological groups of bacteria, such as denitrifiers, requires knowledge of the interactions of a large number of medium components and growth conditions. Genetic or evolutionary algorithms (EAs) are heuristic op...

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Stenotrophomonas terrae sp. nov. and Stenotrophomonas humi sp. nov., two nitrate-reducing bacteria isolated from soil

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