In a previous post, I discussed isotopes and two types of data being collected that utilize isotope ratios – uptake tracer experiments and natural abundance. I spoke at length about uptake tracer experiments (to refresh your memory, N-15 labeled ammonium and nitrate are injected into seawater samples, left to incubate for a specific amount of time, and then analyzed by a mass spec to determine the mass profile of the phytoplankton – giving us a rate of nutrient uptake into the phytoplankton’s biomass). Today’s post is dedicated to natural abundance nitrogen isotope ratios, which looks at the naturally-occurring isotope ratios in the ocean.
To understand natural abundance, it is first essential to understand how different isotopes react. The majority of the processes I’ve discussed over the past three weeks are enzyme-mediated. Enzymes are biologic catalysts and lower the energy needed for a reaction to occur, so the substrate (what a reactant is called when enzymes are used) binds to an enzyme and then the reaction occurs to give a product. The graphic below was designed by my brother and depicts the marine nitrogen cycle, including the conversions among all of the different compounds, the enzymes involved (written in italics) and the fractionation factors (the numbers, discussed below) associated with each.
For natural abundance nitrogen, nitrogen compounds (nitrate and ammonium, for example) act as the substrates. The reactions will occur with both N-14 and N-15 isotopes, but at different rates. The lighter isotope tends to react faster, though the difference is very small. Lighter compounds reacting/moving faster is actually a common theme in chemistry. In gases, for example, lighter gases diffuse (spread) faster while heavier gases diffuse more slowly. This is why helium balloons deflate more quickly than ones blown up manually – the smaller, lighter helium moves around more quickly and is more easily able to escape the balloon due its mass and size.
Back to natural abundance – the difference in reaction rates of different isotopes is called isotope fractionation and determines the 15N/14N isotope ratio. We use this isotope fractionation as an identification agent (almost like a signature) for different processes because the different fractionations that occur result in different characteristic isotope ratios for each process and each nitrogen compound.
Nitrate is actually heavier on average than ammonium, meaning that nitrate has more 15N relative to 14N than ammonium. This is because of additional fractionation of nitrate when it reacts in a process called denitrification. In denitrification, bacteria take nitrate and produce nitrogen gas (N2) that escapes and is lost to the system. Because, as I previously mentioned, the 14NO3– will react faster than the 15NO3–, more 15NO3– is left over.
Nitrate and ammonium are used by phytoplankton to build biomass, and make organic N. Ammonium concentrations tend to be too low to determine a reliable natural abundance isotope ratio. To measure natural abundance nitrate and organic N in phytoplankton, the isotopes are measured via mass spectrometry. To do this, these compounds are converted to nitrous oxide (N2O, which is a much smaller component of the atmosphere) using a “Denitrifier Method” (Sigman, et al. 2001).
Organic nitrogen is first reacted with persulfate in a chemically-mediated process to make nitrate. This nitrate is then used by bacteria to make N2O in a biologically-mediated process (this is Dr. Sigman’s denitrifier method). The N2O is analyzed by mass spec to determine the isotope ratio. The nitrogen passes from the organic nitrogen (from phytoplankton) to nitrate to nitrous oxide so the only nitrogen masses measured are those from the phytoplankton – nothing else is introduced into the reaction.
Studying natural abundance is important, as Dr. Fawcett mentioned in her spotlight, because knowing whether phytoplankton consume ammonium or nitrate helps determine their effect on the carbon cycle and climate. Nitrate is a net remover of atmospheric carbon dioxide while ammonium has no net effect on CO2.
Until next time,
On a quick personal note, I want to wish my dad a very happy birthday! Miss you and love you! XO