Effect of different carbon substrates on nitrate stable isotope fractionation during microbial denitrification

In batch experiments, we studied the isotope fractionation in N and O of dissolved nitrate during dentrification. Denitrifying strains Thauera aromatica and "Aromatoleum aromaticum strain EbN1" were grown under strictly anaerobic conditions with acetate, benzoate, and toluene as carbon sources. ¹⁸O-labeled water and ¹⁸O-labeled nitrite were added to the microcosm experiments to study the effect of putative backward reactions of nitrite to nitrate on the stable isotope fractionation. We found no evidence for a reverse reaction. Significant variations of the stable isotope enrichment factor ε were observed depending on the type of carbon source used. For toluene (ε¹⁵N, -18.1 ± 0.6‰ to -7.3 ± 1.4‰; ε¹⁸O, -16.5 ± 0.6‰ to -16.1 ± 1.5‰) and benzoate (ε¹⁵N, -18.9 ± 1.3‰; ε¹⁸O, -15.9 ± 1.1‰) less negative isotope enrichment factors were calculated compared to those derived from acetate (ε¹⁵N, -23.5 ± 1.9‰ to -22.1 ± 0.8‰; ε¹⁸O, -23.7 ± 1.8‰ to -19.9 ± 0.8‰). The observed isotope effects did not depend on the growth kinetics which were similar for the three types of electron donors. We suggest that different carbon sources change the observed isotope enrichment factors by changing the relative kinetics of nitrate transport across the cell wall compared to the kinetics of the intracellular nitrate reduction step of microbial denitrification.