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.

Nitrogenase Activity and Temperature

While being here in Iceland, Jackie and I both have a great opportunity to develop a research project.  Over the past couple of weeks I have noticed that particular species of nitrogen fixers are growing in select stream temperatures.  For example, “Rock” Nostoc -  Nostoc c.f. pruniforme (Kützing) Hariot, is only found in colder stream temperatures, while “Pink” Nostoc, - Nostoc spongiaeforme Agardh ex Born Flah, is found in warmer streams.  It was interesting to see such a distinct species preference to temperature.  I started to wonder what would happen to these species if the stream temperature that they are acclimated to were to change.

"Rock" Nostoc - Nostoc c.f. pruniforme

            It is well documented that global temperatures are increasing (NASA 2013). All organisms, in general, have several physiological processes that are regulated by temperature-dependent enzymes including cellular respiration, photosynthesis, and for a special group, nitrogen fixation.  Enzymes have a threshold for both cold and hot temperatures. As Jackie mentioned in the previous post, The Number, a select group of organisms have the ability to obtain and use nitrogen gas from the atmosphere, which is unavailable to non-nitrogen-fixers. These organisms are called cyanobacteria and they have a specific enzyme, called nitrogenase, which allows them to do this.  Nitrogenase functions to break the triple bond in nitrogen gas to yield ammonium, which is then used by the cyanobacteria to build biomolecules essential for growth.  Fixing nitrogen is an energetically expensive process and, therefore, not advantageous in nitrogen-rich environments.  However, the streams we are working in are not nitrogen rich, and many species of cyanobacteria are definitely present, with some clear shifts in species composition across the temperature gradient.  Given these observations, I began to wonder, how does temperature affect nitrogenase activity in different aquatic cyanobacteria species?  Is the rate strictly driven by temperature, or have these species adapted to certain stream temperatures in ways that lead to differing relationships between temperature and nitrogen fixation rates among the different species? Can cyanobacteria found inhabiting cold streams rapidly increase nitrogen fixation rates in warmer streams and vice versa?

            In order to investigate this question, we will be doing a reciprocal transplant experiment. We will collect dominant cyanobacteria (mostly Nostoc pruniforme) found in cold streams (~10˚C) and transplant them into both colder (~5˚C), warmer (15˚C), and hot (25˚C) streams. We will do the same for Pink Nostoc - Nostoc spongiaeforme, whose resident mean stream temperature is about 15˚C, as well as other dominant cyanobacteria species.  By the end of the transplants, cyanobacteria from each of their resident streams will be relocated to other streams spanning this temperature gradient, with 5-6 replicates for each species. I know I mentioned previously, that temperatures are increasing, so why put samples into colder temperatures?  Assuming nitrogenase reacts to temperature like other enzymes, its activity rate should decrease in colder temperatures and increase in warmer temperatures, up to some threshold.  Placing the dominant cyanobacteria species across a wide temperature gradient and measuring their nitrogen fixation rates will help us to better interpret and understand the relationship between temperature and enzymatic activity and where and under what conditions we should find each species.

          

"Pink" Nostoc - Nostoc spongiaeforme

  The goal of this experiment will be to see how these different species respond to different temperatures, and their ability to acclimate to a new environment. It is important to know the temperature threshold these species can withstand and how nitrogen fixation rates are likely to change in a warming world. If their temperature threshold is limited, it might be possible that in coming years, community composition will shift due to rising temperatures which could lead to species being out competed or lost entirely. Nitrogen-fixers play an important role in ecosystems where nitrogen is limited because they provide a source of nitrogen for other organisms, which feeds back on the ecosystem as a whole by influencing photosynthetic rates, the production of invertebrates and fish, and how other elements like carbon and phosphorus cycle as well.

"The" Number

We found algae!

We finally had some decent weather after days of rain and wind, and we were able to go out and make our first measurements of nitrogen fixation on the tiles associated with the channel temperature experiment. Algae (and all photosynthetic organisms) typically acquire their source of nitrogen from the soil and water around them, but some species, called cyanobacteria, are able to acquire it from the atmosphere. The species that can do this have an enzyme called nitrogenase that can break apart the two nitrogen molecules in nitrogen gas (N2), the most abundant gas in the atmosphere. They then use the nitrogen to build amino acids and other nitrogen containing molecules for growth. 



This summer we are planning to measure nitrogen fixation with three different approaches as a comparison of the methods, as well as to use them as a check against each other.  For

Chemical conversion of nitrogen fixation (left) and
how it compares to acetylene reduction (right).

our first sampling day, we only used one method, which is called the Acetylene Reduction Assay, or ARA. The ARA method involves making acetylene gas, which we do in the field using calcium carbide and water in a flask, and then collect the gas in either a balloon or syringe. We then inject the acetylene gas into gas-tight chambers with the algal samples and then take an initial and final sample of the gas in the chamber. During the incubation the algae in the chamber are breaking the triple bond between the two carbon atoms in acetylene to produce ethylene (see diagram), which is similar to the chemical reaction in nitrogen fixation (see diagram), and we are able to measure production of ethylene gas and use this as a measure of how much nitrogen the algae are fixing. 



Working on getting "the" number.

Over the years, the ARA method has been modified and refined for better results while still trying to keep the procedure fairly simple. This summer, we are collaborating with another project and we are measuring nitrogen fixation on tiles that have been colonized with algae.  As part of this effort, we had to modify the ARA method again for use with custom chambers that were built for the project. This came with several challenges that involved some creative thinking and problem-solving skills, which are essential skills to have when conducting research. The physical act of research is easy; going out in the field, collecting data using technical instruments, putting samples of water and algae into vials, and following previously thought out methods.  However, it is naive to think that’s all there is to research. As Jill always says, “It’s easy to get 'a' number, but more challenging to get 'the' number”. What she means is that it’s easy to go out into the field, spends hours collecting data, and reading numbers off of instruments, but the hard part is deciding what that number tells us, and if we used correct and precise methods for obtaining the actual number that represents part of the answer to our question. For example, when we are trying to figure out how to make gas-tight chambers we have to think like a gas molecule and really channel our inner Sherlock Holmes to make sure we are getting good data, but at the same time not be biased.

So far, our research has been really frustrating, impossibly complicated, and infinitely

Excited after a day in the field of
hiking and looking at algae.

rewarding. I love a good challenge and this research is important in helping us understand how whole ecosystems work, which we can then use to help gauge how they are going to be affected by human activity. There is still so much we don’t know about our world and the ecosystem services we depend on for survival. I am starting to see how easy it is to build an entire career’s worth of work in research. I feel invested and driven by curiosity and I am really looking forward to seeing where it takes me.

"Re" Search

The last couple of days have served as a great reminder that when a problem arises with a critical part of an experiment, rarely is there a single solution. I thought that when an experiment was about to begin, all of the details were worked out, and the only tasks left were to collect data and analyze it.  Wrong. It has been said on several occasions that “once you smooth out the kinks, data collection is a breeze”. Finding the kinks is easy; it’s the solution that’s tricky.

The 300mL chamber that will be used
 to measure nitrogen fixation

One of our methods requires the use of an air-tight chamber. On the surface, it sounds like an easy task. Only once you try to actually make a chamber air tight do you realize the difficulty.  In our collaboration with Tanner Williamson, we are using chambers that have been designed especially for this experiment. These chambers hold only about 300 mL of water, which is ideal for measuring biological processes on a small amount of algae. The small chamber volume is important because the tiles that we are measuring nitrogen fixation on are barely a cubic inch in size. The added bonus of these chambers is that it has a recirculating fan that circulates the water within the chamber and mimic stream movement. The double-edged sword is that these chambers only have one opening. The chambers that were used in our work last year had two openings; however, those chambers were much larger (2 liters) which makes measuring nitrogen fixation more difficult. The benefit of having two openings comes into play when we are required to simultaneously add gas and remove water using two different ports. These new chambers posed two problems: being air-tight and only having one opening.

Deciding to address the gas-tight issue first, we attempted to cap the opening of the chamber with a rubber septum. It turns out that this is extremely hard to do, as you are essentially forcing an object against a positive pressure gradient. Even though we managed to get the septum on, the chambers were over-pressurized and some airspace remained inside the chamber. Plan B involved a simpler method. Underwater there should be no air, thus removing the problem of trapping air inside the chamber and over-pressurizing. Placing the septum over the top of the chamber while the septum and chamber are submerged creates a chamber that is free of air.

Using a chamber with only one opening has a few challenges within it. We are required to add gas and remove water at the same time for one of our methods. This technique becomes difficult to do when the only way into the chamber is through the septum at the top (about the size of a quarter). Two insertions (one for the gas, the other for the water) requires two people. We also have to insure that we are not pulling out the newly injected gas as we are removing water from the chamber. We then had to address the issue of which insertion would work best for injecting the gas. In other words, should we inject the gas at the top of the chamber or at the bottom? To answer this, we added blue food coloring to the chamber, with the fan running, and observed the flow pattern of the water. The food coloring instantly moves toward the fan and begins to dilute.  Based on this, we decided it would be most beneficial to add the gas at the top of the chamber and pull the water out from the bottom.
     Several hours of critical thinking mixed with trial and error resulted in these problems being solved. At the end of the day, it is extremely gratifying to have created a solution to a problem that at the beginning of the day seemed unfix-able.   These challenges have helped me to understand that critical thinking and creativity go hand in hand during research.