Control of Denitrification in a Permanently Ice-Covered Antarctic Lake: Potential for Regulation by Bioactive Metals

Agency: NSF
CoPI: M. L. Wells, University of Maine
Ward Lab Participants: Caroline Tuit

Denitrification is the main loss term for fixed nitrogen from ecosystems, and thus its rate and regulation may directly affect primary production and carbon cycling over short and long time scales. We investigated the role of bioactive metals in regulating denitrification by exploiting a natural experimental system that occurs in a permanently ice-covered lake in the Taylor Valley of East Antarctica. Chemical distributions in the two lobes of Lake Bonney imply that denitrification occurs in one lobe but not the other. Most of the obvious biological and chemical variables that usually influence denitrification, and might account for the difference between the two lobes, have been ruled out by previous study. Previous research has also demonstrated that denitrifying bacteria are present in both lobes of the lake and isolates derived from the lake show temperature and salinity optima consistent with their persistence in this environment.

Field Work at Lake Bonney
In field seasons in 1999 and 2000, we carried out a number of experiments at Lake Bonney in an attempt to resolve the mystery.

-- Total metal concentrations and metal speciation were determined in depth profiles of both lobes, focusing mainly on the suboxic zone of both lobes of Lake Bonney. In general, metal concentration s were high and increased dramatically in the suboxic regions. Silver concentrations were significantly higher in the East lobe, while copper concentrations were much lower in the East, compared with the West.  We hypothesize that a specific Ag/Cu interaction may be involved in inhibition of denitrification in the East Lobe of the lake.

-- Manipulative experiments in which the bioactive metal availability of lake water was altered in attempts to induce denitrification in incubations with water in which it appears to be inhibited were carried out in gas tight trace metal clean incubation bags. Both total bacterial activity (thymidine incorporation) and denitrification (acetylene block) rates were measured. The distribution of bacterial activity in both lobes has been measured many times in the past, and as expected, rates declined below the chemocline. Denitrification was detected in the West lobe with a maximum at 16 m, at the oxic/anoxic interface. Bacterial activity and denitrification were both detected in the East Lobe, but activity was extremely low below the chemocline. Denitrification was detected in much deeper water, in the vicinity of the nitrous oxide peak at 25 m. Chelators had no consistent effect on either total activity or denitrification.

The next phase of the project involves both lab and field work in 2004-2005. Laboratory experiments will investigate the interactions between copper and silver in the function of denitrification enzymes. In the next two field seasons we will employ a suite of physiological indicators (vital stains with flow cytometry, NO analysis) and manipulative experiments with metal availability to try to identify exactly what environmental factors maintain microbial life at the edge of viability in the Lake Bonney.

Laboratory Culture Experiments
We also performed experiments on cultivated denitrifying bacteria, including isolates obtained previously from Lake Bonney, in order to investigate the trace metal tolerances and requirements for denitrification. Denitrifying bacteria could be easily limited for both iron and copper. Copper limitation had dramatic effects, causing the accumulation of either nitrite or nitrous oxide in the growth medium. Iron limitation caused a reduction in overall denitrification rate, but copper had specific effects on specific enzymes (NiRK type nitrite reductase and nitrous oxide reductase) that require copper as a cofactor. (Granger and Ward, 2002).

Cultures of Pseudomonas stutzeri grown in the presence of sufficient or limiting Copper concentrations.
open triangles: NO₂ in Copper replete cultures
closed triangles: NO₂ in Copper limited cultures
open circles: N₂O in Copper replete cultures
closed circles: N₂O in Copper limited cultures