The TriMethod Tournament

When using 3 methods to
estimate nitrogen fixation, time and
organization of the essence -
and I made sure to keep us on task.

Although it did not involve dragons, mermaids, and hedge mazes full of deadly creatures, we, like Harry Potter in the TriWizard tournament, embarked on a mission that verged on the impossible. It would require skill, perseverance, tenacity, and a little bit of luck.  We have taken it upon ourselves to use not just one, but three different methods in the field to measure nitrogen fixation, which can be intense, especially when dealing with all of them in one day and having to multitask and run them simultaneously. It took all of our focus, along with organization on everyone’s part, to be able to run this smoothly.  Luckily, we had a great team of researchers that worked really well together. I took on the roll of time keeper and task manager and made sure everyone knew what they were doing at which time, because we had time sensitive incubations that were running simultaneously, so there were many things going on at once.

Here, Jill and I are adding 15N2 gas (contained in
the small gray cylinder) to our chambers containing
algae from the different temperature treatments.

Along with the Acetylene Reduction Method (ARA), which I mentioned in a previous post, we were also using a 15N2 method, and a relatively new method called Membrane Inlet Mass Spectrometry (MIMS). All of these methods are a way in which we can measure nitrogen fixation. Unlike the ARA method, which indirectly estimates the rate at which the algae are able to break apart nitrogen gas molecules, the 15N2 method measures the rate at which those nitrogen gas molecules are being incorporated into the biomass of the algae, or, in other words, how much nitrogen did the algae consume and assimilate. In the natural world, there are different “types” of nitrogen atoms, which we call isotopes, but they are exactly the same in every way except they vary slightly in weight. For example, there are 14N and 15N atoms, and in the natural world 14N is much more common in the atmosphere, making it is very hard to track.  In comparison, 15N atoms are very rare, so if we provide a large source of 15N to the nitrogen fixers and they use them, we can use the 15N as a tracer, and track how much nitrogen the algae incorporated into their biomass. So, just like in the ARA method where we inject acetylene into gas tight chambers with algal samples, in the 15N2 method we inject a known amount of 15N2 gas into the chambers instead and allow the algae to incubate for 2 hours to take up this added nitrogen gas where all of the nitrogen atoms are the heavier, and more rare, 15N. However, unlike the ARA method where we collect a gas sample from the chamber at the end, we will directly take the algae from the chamber and use specialized equipment to analyze it for the 15N tracer that we added to the chamber and see how much of it is now in the algae.

It is definitely a coordinated team effort to get all of our
 various chambers set up and running - and in the rain!!

The third method we are using, MIMS, is much simpler in theory, and more direct, than the other methods.   However, the technology associated with this method has only recently become available and it is still very new.  Essentially, we take a water sample from the beginning and the end of the 2 hour incubation with our algal samples. We then preserve the water sample and its associated dissolved gases, which includes N2, and then send it off to be analyzed on a Membrane Inlet Mass Spectrometer (MIMS), which will tell us how many N2 molecules were in the water at the beginning and how many there are at the end. At the end of the incubation, there should be fewer molecules in the water if nitrogen fixation is occurring, as those N2 gas molecules get taken up and incorporated into the algal biomass in the form of proteins and other biomolecules essential for growth.

It's working!  Yes!

So at the end of the day we had spent 10 hours in the field and we were exhausted, but excited to have completed an exceptionally successful day in the field. At the end of the day, I found it to be very satisfying to know that everything that we worked so hard on up to this day had paid off.  And, just wait until you see the data - they are very exciting!!  But, just like the journey of Harry Potter, not all can be revealed at once.  You must wait until the full story unfolds...all in good time.