N-215. Development of a System to Quantify Denitrifier Gene Expression in Agricultural Soil and to Study Its Relation to Nitrous Oxide Flux and Denitrification

S. L. Henderson1, C. E. Dandie2, C. Goyer2, C. Patten1, B. J. Zebarth2, D. L. Burton3, J. T. Trevors4;
1Univ. of New Brunswick, Fredericton, NB, CANADA, 2Agriculture and Agrifood Canada, Fredericton, NB, CANADA, 3Nova Scotia Agricultural Coll., Truro, NS, CANADA, 4Univ. of Guelph, Guelph, ON, CANADA.

There is a gap in the understanding of how denitrifier community abundance and activity correlate with denitrification and nitrous oxide (N2O) emissions in soil. As part of a study to address this gap, the objective was to develop a system for quantifying denitrifier community abundance and activity and to investigate their relationship with denitrification. Using this system we can test the hypothesis that an increase in denitrification is due to an increase in gene expression and/or abundance of the denitrification community. Agricultural soil was supplemented with 500 mg KNO3-N kg-1 (to ensure nitrate was non-limiting) and either 0 or 500 mg glucose-C kg-1. The treated soil was placed in sealed jars, C2H2 added, and kept at 4oC for 16 h to ensure C2H2 diffusion into the cores. Cores were then incubated at 25oC over 48 h. Soil was destructively sampled over 48 h and nucleic acids were extracted. Quantitative PCR (Real-Time PCR) was used to quantify the number and activity of the nosZ gene bearing denitrifiers and Pseudomonas mandelii and closely related species (nirSP) communities. Denitrification (N2O production in the presence of C2H2) and concentration of nitrate and extractable organic carbon were measured. The abundance of the nosZ containing community, average of 2.0 x 107 gene copies g-1 dry soil, was not affected by carbon treatment whereas glucose addition resulted in a 20-fold increase in abundance of the nirSP community. Surprisingly, gene expression of the nosZ containing denitrifiers and nirSp did not change over time or treatments (1 x 104 and 1 x 106 transcripts g-1 dry soil, respectively), suggesting that gene expression had already reached its maximum when 25oC incubation began. Since the induction of gene expression was not detected, the gene expression is inconclusive for addressing the hypothesis. Subsequently, the system was modified to reduce time between treatment addition and incubation at 25oC to less than 15 min. Preliminary results showed an induction of gene expression at 4 h for nosZ and 8 h for nirSP suggesting that the modified system is better suited to study the link between gene expression and denitrification.