Culture Studies of Nitrogen and Oxygen Isotope Effects Associated with Nitrate Assimilation and Denitrification

The isotope effects of nitrate-consuming reactions such as nitrate assimilation and denitrification are potential indicators of the physiological state of the organisms carrying out these reactions. Moreover, an understanding of these isotope effects is needed to use the stable isotopes to investigate the fluxes associated with these reactions in modern and ancient environments. We have used batch cultures to investigate the nitrogen (N) and oxygen (O) isotope effects of (1) nitrate assimilation by eukaryotic and prokaryotic algae and by heterotrophic bacteria, and (2) nitrate reduction by denitrifying bacteria. We observe intra- and inter-specific variation in isotope effect amplitudes and, in the case of denitrifiers, indications of isotope effect decreases during individual nitrate drawdown experiments. However, the measured N and O isotope effect ratio is close to 1 for all studied organisms, with the exception of an unusual denitrifier (Rhodobacter sphaeroides) that possesses only periplasmic (non-respiratory) nitrate reductase. This observation and other findings are consistent with nitrate reductase being the predominant source of isotope fractionation and with most isotope effect amplitude variability arising from variable degrees to which nitrate imported into the cell is reduced versus effluxed back into the environment; the more efflux, the more complete the expression of the fractionation imparted by nitrate reduction. If this is the case, then isotope effect amplitudes in the field should be related to physiological conditions in the environment, a prediction that, we argue, is supported by recent studies of (1) nitrate assimilation in the polar ocean and (2) denitrification in sediment porewaters.