Award: OCE-1233897

In the ocean, denitrifying bacteria effectively "breathe" nitrate (NO3-) when dissolved oxygen is absent in the water, ultimately converting nitrate into N2 gas. This process is important because it essentially removes nitrate nutrient rom the ocean, which has consequences for the fertility of the surface ocean. One way to determine how much of the nitrate in the ocean is denitrified by bacteria is by constructing a nitrogen isotope mass balance. Nitrogen has two naturally occurring stable isotopes, 14N and 15N, that react at slightly different rates during biological transformations. Nitrate with 14N is denitrified to N2 at a slightly faster pace than 15N nitrate. By measuring the relative abundance of 15N/14N in ocean nitrate, we can make deductions about how much denitrification occurred relative to nitrogen input into the biosphere from biological nitrogen fixation. An important parameter that is needed to construct the mass balance is the so-called "isotope effect" imparted on nitrate by denitrifiers, which is a measure of the relative rates of reaction of 15NO3- vs. 14NO3-. In this study, we investigated which factors can modulate the nitrogen (N) isotope effect associated with denitrification in laboratory cultures of bacterial denitrifiers. We determined that the carbon substrate on which the bacteria are cultured does not modulate the N isotope effect consistently. Contrary to previous findings, the cellular nitrate reduction rate of cultures is not correlated to the isotope effect. Indeed, while the cellular reduction rate increases up to 10 fold during growth of the culture, the isotope effect is unchanging throughout. The increase in nitrate reduction rate is matched by a coincident increase in the activity of the respiratory nitrate reductase, NAR, which suggests that nitrate uptake and reduction are co-regulated, explaining the unchanging isotope effect. The isotope effect did change when cultures were purged with either trace oxygen or with nitrous oxide gas. We hypothesize that this occurs because both oxygen and nitrous oxide reduce nitrate transport into the cell, which causes a reduction in the denitrification isotope effect. In all, our findings suggest that the denitrification isotope effect is variable, subject to conditions that influence nitrate transport into the cell. We also investigated whether the isotope effect of the nitrate reductase enzyme, NAR, can be modulated as a function of experimental conditions. We determined that the enzymatic isotope effect of NAR is unchanging when enzyme activity is fuelled by the non-specific reductant methyl viologen, but that the isotope effect can vary when enzyme activity is fuelled by hydroquinone, its native reductant. Thus, we hypothesize the organism-level isotope effect can change due to changes in the enzymatic isotope effect, in response to cellular reductant concentrations. Last Modified: 10/05/2015 Submitted by: Julie Granger