Measuring Nitrogen Fixation: The Acetylene Reduction Assay

Bree and Abbi taking gas samples.

There are often multiple answers to the same question.  Whether or not those answers are parallel and agree with one another is often a different story. We are interested in determining the rate of nitrogen fixation in stream ecosystems and understanding how rates are affected by temperature. The Hengill Valley in Iceland provides us with a unique natural ecological laboratory to ask our questions, with streams that span across a 25 degree Celsius temperature gradient, while maintaining similar flow, light, and water chemistry. However, what is "the true" rate?  Getting this answer is harder than it may first appear.

Nitrogen fixation is a process completed by cyanobacteria.  While they are the only group of organisms capable of this process, they are found all over the world, in water, soil, and sometimes in association with plants (e.g., legumes). Our work in Iceland focuses on species of algae in streams that fix nitrogen. These organisms have a competitive advantage in nitrogen poor environments, including our study streams where stream water is quite nitrogen poor. They are able to take nitrogen gas, which makes up about 78% of the atmosphere, and convert it (by the use of an enzyme known as nitrogenase) to a biologically available form of nitrogen, which is an important building block for amino acids, proteins, and many cellular processes! Think of it this way: if you needed ice cream, but there was none available, you could turn to cream, sugar, and ice and make your own, if you could get the ingredients and had the right equipment to combine them. This is what cyanobacteria do in order to make proteins - they access nitrogen from the air when other sources are not available.  Evolutionarily genius!

Measuring nitrogen fixation rates on stream 11 -
 the water is super cold!

So you may be thinking, “how on Earth can you figure out how much nitrogen gas is being taken from the atmosphere and converted into the biomass of these nitrogen fixing organisms?” Do not worry - I asked the same question.  In fact, I have learned that there are currently three methods that are used to determine the rate of nitrogen fixation. We have started to answer our question with a method known as the acetylene reduction assay. Now before you panic, let me break it down. This method uses acetylene gas as a "stand in" for nitrogen gas.  Recall that nitrogen gas comprises 78% of the atmosphere.  It is typically difficult to measure the uptake of such an abundant substance. We are fortunate, however, that the nitrogenase enzyme also reacts with acetylene gas (acetylene and nitrogen gas have similar triple-bonded molecular structures), which allows us to use it to gain an indirect estimate of nitrogen fixation. 

The video below shows me preparing acetylene gas filled balloons that we insert into a gas tight chamber.  The balloon is then popped to allow the gas to be readily available to the cyanobacteria.  It is important that we shake the chamber to dissolve the acetylene gas in the water. We take gas samples before and after an incubation period of 2 hours. The gas samples are  then run on a gas chromatograph (also in video), which is used to quantify the amount of different gases in the sample. If nitrogen fixation is occurring in the chamber, the concentration of acetylene will decrease while ethylene will increase, which is the gas by-product created from our acetylene when nitrogenase is active. However, there are also two more methods that we are using to measure nitrogen fixation rates.  How will the results from those methods compare to the acetylene reduction assay?  We can't wait to find out! If you have any questions, please leave us a comment, we would love to talk more about nitrogen fixation. More methods to come next week!