Effect of Different Carbon Substrates on Nitrate Stable Isotope Fractionation During Microbial Denitrification

Abstract

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.

Isotopic overprinting of nitrification on denitrification as a ubiquitous and unifying feature of environmental nitrogen cycling

“…Although many of the important source terms and isotope effects of the N cycle are constrained, some remain equivocal. In particular, recent observations emerging from bacterial and archaeal cultures and from incubations of environmental samples have uncovered isotopic discrimination trends for NO 3 − isotopes that appear at odds with trends typically ascribed to analogous biological transformations in soils and aquifers (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14). This development has led to conflicting environmental interpretations, reflecting a lack of consensus on fundamental isotope systematics of the processes driving the N cycle.…”

Nitrogen isotope dynamics and fractionation during sedimentary denitrification in Boknis Eck, Baltic Sea

“…Nitrite exchanges oxygen atoms with ambient water, so any reoxidation should be visible in oxygen atoms in nitrite. Different recent studies (Knöller et al, 2011;Wunderlich et al, 2012) closely investigated the potential for nitrite reoxidation, and concur that there is no re-oxidation of nitrite during dissimilatory nitrate reduction within bacterial cells under anoxic conditions.…”

“…Accordingly, variations in enzyme equipment between different strains of denitrifiers, in marine and freshwater communities (Wunderlich et al, 2012), still seem a likely explanation of the variant ε 18 /ε 15 ratio.…”

“…During denitrification, the first step is the uptake of nitrate across the cell wall into the cytoplasm, because the respiratory nitrate reductase Nar, which catalyzes the first step of denitrification, is an integral membrane protein with its active site directed towards the cytoplasm. After reduction, nitrite has to be transported out of the cell into the periplasm, where the nitrite reductase (Nir) is located (Wunderlich et al, 2012;Zumft, 1997). Additionally, denitrifiers can also possess a second nitrate reducing enzyme, the periplasmic Nap, but this enzyme is not involved in energy conservation, as it does not translocate protons across the inner membrane.…”

“…6), and the loss rate of nitrate back out of the cell is assumed to be much lower (i.e., k 1 k −1 , cf. Granger et al, 2008;Wunderlich et al, 2012). Within the cytoplasm, nitrate is reduced by Nar at rate k 2 , assumingly with an isotope effect of ∼ 19 ‰.…”