Seasonal Variability of Cultivable Nitrate-Reducing and Denitrifying Bacteria and Functional Gene Copy Number in Fresh Water Lake

Abstract

This study describes the seasonal course of denitrifying and nitrate-reducing bacteria in a dimictic mesotrophic lake (Lake Scharmützelsee, Brandenburg, Germany) within a three-year period from 2011 to 2013. The bacterial cell numbers were quantified by the fluorescence microscopy, most probable number (MPN) and PCR-dependent quantification of the chromosomal 16S rDNA and of the nirS and nirK gene copy number. The highest seasonal differences (up to three orders of magnitudes) have been measured using MPN in the epilimnion. This variation was not reflected by PCR-dependent approaches or direct microscopical enumeration. At adverse conditions (low temperature and/or low nitrate concentrations), the differences between MPN and gene copy numbers increased by up to five orders of magnitudes and decreased to one magnitude at favourable environmental conditions. These results can be explained best by an increasing ratio of viable but not cultivable (VBNC) cells or dead cells at impairing conditions. In the hypolimnion, the courses of MPN and nir gene copy numbers were similar. This can be explained by a higher feeding pressure and therefore smaller amounts of dormant cells. In the pelagial in general, the total cell numbers enumerated by either microscopical or molecular approaches were similar. In the sediment, more than 99% of the DNA was obviously not related to viable bacteria but was rather DNA in dead cells or adsorbed to particle surfaces.

Keywords: MPN; PCR; denitrification; dimictic lake; epilimnion; hypolimnion; microscopical cell number; nitrite reductase; sediment; vbnc state.

Figure 1

Courses of temperature (a), oxygen (b) and nitrate concentration (c) in the epilimnion (grey line) and hypolimnion (black line) of lake Scharmützelsee from 2012 to 2013.

Figure 2

Courses of nitrate concentration and cell numbers in the epilimnion of lake Scharmützelsee from 2012 to 2014: (a) microscopic cell counting (), 16S rDNA gene copy number (), (b) MPN of nitrate-reducing bacteria () and denitrifying bacteria (), sum of nirS and nirK (), nitrite concentration (grey line).

Figure 3

Combined annual courses of cell numbers and nitrate concentrations in the epilimnion of lake Scharmützelsee: (a) microscopic cell counts (), 16S rDNA cell-number estimation (), temperature 2011 (solid grey line), 2012 (dashed black line), 2013 (dotted black line) (b) numbers of denitrifying bacteria estimated by the sum of nirS and nirK gene copies (), MPN of nitrate-reducing bacteria () and denitrifying bacteria (), (c) difference between log10 transformed cell numbers (nir)and MPN of denitrifying bacteria (+), nitrate concentration 2011 (solid grey line), 2012 (dashed black line), 2013 (dotted black line).

Figure 4

Courses of nitrate concentration and cell numbers in the hypolimnion of lake Scharmützelsee from 2012 to 2014: (a) microscopic cell counting (), 16S rDNA cell-number estimation (), (b) number of denitrifying bacteria estimated by the sum of nirS and nirK gene copies (), MPN of nitrite-reducing bacteria (()) and denitrifying bacteria (), nitrate concentration (black line).

Figure 5

Combined annual courses of cell numbers and nitrate concentrations in the hypolimnion of lake Scharmützelsee: (a) 16S rDNA cell-number estimation (), number of denitrifying bacteria estimated by the sum of nirS and nirK gene copies (), MPN of nitrate-reducing bacteria () and denitrifying bacteria (), nitrate concentration 2011 (solid grey line), 2012 (dashed black line), 2013 (dotted black line), (b) difference between log10-transformed cell numbers (nir) and MPN of denitrifying bacteria (+).

Figure 6

Courses of nitrate concentration in the hypolimnion and cell numbers in the upper sediment layer of lake Scharmützelsee from 2012 to 2014: microscopic cell counting (), 16S rDNA cell-number estimation (), number of denitrifying bacteria estimated by the sum of nirS and nirK gene copies (), MPN of nitrite-reducing bacteria () and denitrifying bacteria (), nitrate concentration (black line).