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

TLDR

The study aimed to identify optimal isolation conditions for denitrifying bacteria by evaluating medium parameters. An evolutionary algorithm was employed to analyze the complex interactions among medium parameters and their effects on the number and diversity of isolated denitrifiers. Optimal media (pH 7, 3 mM nitrogen, 1 ml vitamin, no NaCl or riboflavin, ethanol or succinate as carbon source, C/N ratio 2.5–25) were identified, yielding three highly suitable formulations; 199 denitrifiers were isolated, predominantly Betaproteobacteria (50.4 %) and Alphaproteobacteria (36.8 %), with additional Gammaproteobacteria, Epsilonproteobacteria, Firmicutes, and Bacteroidetes, demonstrating a more diverse community than previously reported.

Abstract

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.