Direct detection of denitrifying bacteria in groundwater by GeneFISH

Abstract

Denitrification, nitrate reduction, is a key microbial metabolism that proceeds under the low oxygen condition in soil, sediment, and various subsurface environments. It is suggested from in vitro experiment that denitrification decreases redox potential in a given environment as groundwater, leading to changes in behavior of migration of radionuclides. Thus, to elucidate the abundance of denitrifying bacteria in a given groundwater is regarded as an essential information in understanding the migration of radionuclides in groundwater. Fluorescence in situ hybridization (FISH) technique is a useful tool to elucidate the abundance of phylogenetically selected groups of bacteria, but bacteria, being capable of denitrification, distribute widely in phylogenetic groups. To detect denitrifying bacteria, we need to find them by their functional genes. GeneFISH technique detects the target bacteria by their functional genes directly, but it is not easy to apply this technique to natural bacterial community because of the difficulty to insert a large molecule to the target bacterial cell. We tried to enhance the insertion of large molecule of gene probe into the target cell through modifying the combinations of employed enzymes and found a good treatment procedure to detect dominant denitrifying bacteria in the examined groundwater with high efficiency.